Bacterium detector

ABSTRACT

An object of the present invention is to offer a microorganism-detecting apparatus where the microorganism causing a food poisoning can be detected/identified by a simple operation and also a disposal treatment can be conducted safely and surely. An apparatus for achieving the object is as follows: The apparatus for detecting microorganism as mentioned below is portable and enables one to selectively incubate and detect the microorganism (particularly those which are a cause of food poisoning) without skillfulness and the detecting apparatus containing pathogenic microorganism (such as that which is a cause of food poisoning) can be safely and surely disposed. The apparatus is characterized in having at least (a) a container ( 1 ) for holding a medium which is used for culturing the microorganism to be detected during the period of the incubation of said microorganism; (b) a microorganism-collecting part ( 6 ); (c) a structure ( 3 ) for making possible a process of receiving, in a noncontact manner with the microorganism-collecting part, the medium used for culturing the microorganism to be detected until the incubating stage of the microorganism; and (d) a structure ( 9 ) for disinfecting the medium after incubation.

TECHNICAL FIELD

The present invention relates to an apparatus for selectively detectingmicroorganisms whereby pathogenic microorganisms, particularly foodpoisoning microorganisms and drug-resitant microorganisms such asmethicillin-resistant Staphylococcus aureus (MRSA) can be easily andsimply detected and/or identified. More particularly it relates to anapparatus for a selective detection of microorganisms which isparticularly suitable even for private and domestic use where itsstorage for a long time can be expected, which can be immediately usedat any time when needed, which has a portable form such that it can beconveniently and easily carried, which is capable of safely detectingand identifying a food poisoning microorganism or the like by performinga simple operation, and which can be disposed of safely, surely andeasily after its use.

BACKGROUND ART

Pathogenic microorganisms which are present in the living environmenthave been considerably overcome due to a high development in medicalservice and progress in therapy in recent years, particularly inventionsand developments in antibacterial and antibiotics and, moreover, due toan improvement in hygiene. However, once they break out, big damage andfatal problems may result, and thus pathogenic microorganisms remaincauses of big problems.

Examples of the pathogenic microorganisms which cause troubles whenpresent around us are those causing food poisoning and those resistantto antibiotics, such as methicillin-resistant Staphylococcus aureus(MRSA) and the like. Thus, there are problems such as the occurrence ofa lot of patients infected by these microorganisms, and hospitalinfection as well.

Taking food and beverage or the “eating” action is essential for animalsincluding human beings and, therefore, in spite of progress in scienceand high development in technique in recent years, it is still difficultto completely prevent “food poisoning” caused by taking food andbeverage. On the other hand, prevention of food poisoning as such hasbeen more and more important because of the Product Liability Law whichhas been inaugurated recently.

Food poisoning is roughly classified into three types depending upon itscause, i.e. that by natural poison (toxin) (such as swellfish poison(Tetraodontidae toxin) and mushroom poison), that by putrefaction offood and beverage (such as by decomposed products of food and beverage),and that by contamination by or proliferation of bacteria (such asStaphylococcus, Salmonella and Vibrio parahaemolyticus) in food andbeverage. Among them, food poisoning by natural poison can be easilyavoided by preventing the intake of food/beverage containing the causingpoison. In addition, detection of putrefaction of food/beverage by meansof checking the changes in appearance, smell, etc. is usually easy.Therefore, prevention of food poisoning due to putrefaction offood/beverage is relatively easy in many cases.

In contrast to those two types of food poisoning, contamination by orproliferation of the microorganisms is usually invisible in the case offood poisoning by bacteria (that is, the bacterial food poisoning) and,moreover, detection of this contamination or proliferation is verydifficult without participation of professional people. Accordingly, thebacterial food poisoning is a food poisoning which is most difficult toavoid.

Occurrence of food poisoning associated restaurants, caterers forluncheon and feeding facilities affects are significantly many peopleand, in addition, the symptoms is serious such as severe vomiting,diarrhea, abdominal pain, high fever, etc., whereby it is apt to resultin a particularly serious problem such as stoppage of business of thefood provider for a long term. A further problem is that, since themicroorganism causing such food poisoning produces toxic substances suchas enterotoxin in the body of infected patients, it is necessary toconduct a therapy after infection and, moreover, this therapy is veryoften accompanied by a difficulty.

Accordingly, it is quite necessary that the microorganism which is acause of such a symptom is found at an early stage and that a preventivemeasure such as disinfection is conducted for preventing the poisoning.

As a result of the use of antibacterials and antibiotics, occurrence ofdiseases, especially infectious diseases such as contagious disease, hassignificantly decreased. On the other hand, however, many resistantmicroorganisms have appeared as a secondary phenomenon resulting in abig problem. There is a problem that, when a powerful antibioticsubstance is developed, resistance thereto occurs immediately and, inaddition, hospital infection by such resistant microorganisms havebecome a big problem too. Since patients including those suffering fromsevere disease and aged people has an decreased resistance,microorganisms which are resistant to antibiotics cause a intractableinfectious disease whereby, even when the infection is found, there willbe no effective therapy and that is a big problem. For preventing thehospital infection caused by such resistant microorganisms, it isnecessary to specify the causing microorganism in an early stage ofinfection, to find its presence at an early date and to effectively andappropriately prevent the infection by means of disinfection, etc.Accordingly, it is necessary for hospitals to periodically check whethersuch microorganisms appear in the medical environment.

Up to now, there has been no method other than common preventingmeasures (such as keeping the cooking utensils, e.g. kitchen knife andchopping board, clean and also keeping hand and fingers of the personsparticipating in cooking clean) for preventing the food poisoning. Evenat present, when food poisoning occurs, only an ex posto facto measure(i.e. the causing microorganism is detected and confirmed by a search(by incubation of the suspected microorganism found in thefood/beverage) of the public health center controlling the place wherethe poisoning occurred) is available. In addition, detection andconfirmation of the causing microorganism by the center as such usuallyrequire quite a long period (e.g., around one to two weeks). Further,the search by the public health center needs a “prescribed procedure”and, moreover, in view of honor or reputation (such as an entire loss ofcustomers by “rumor”), such a procedure is not always fully activated.Additionally, it is not always possible to detect and confirm thecausing microorganism so simply.

In the investigation at the professional organizations such as publichealth centers and hospitals, a method which has been adopted is thatvarious samples as mentioned above and those collected from patients andfrom a medical environment are incubated on agar plates using aselective medium, and the colonies formed on the medium are observed bythe naked eye, or the microorganism incubated as such is confirmed byother detecting and confirming measures. Recently, a judging methodusing polymerase chain reaction (PCR) has been adopted as well. However,the operations are troublesome in all of such conventional methods and,in judging the colonies grown on the agar medium, professional techniqueand skillfulness are needed for identification. In addition,professional technique and skillfulness as well as specific devices orthe like are required for the operations from collection of the sampleto its incubation and there is a disadvantage that total cost for thedetection is high. Further, in the case of a method utilizing a PCR, itis again necessary to apply professional technique and skillfulness andthere is another problem that, for certain microorganisms,discrimination of them from nonpathogenic ones is very difficult.

For a device for solving these problems, an apparatus accommodating aliquid medium enclosed in a capsule and a tampon for collectingmicroorganisms in a capsule-container is proposed, for example, inUnexamined Japanese Patent Publication Sho 62-171671 (JP Sho 62-171671A). Because such an apparatus contains pathogenic microorganisms afterincubation, however, it is difficult to discard it. Therefore, in theprior art the used apparatus has been discarded after disinfectingsteps, including opening the used container followed by addition of adisinfectant solution to the medium containing the microorganisms.

Because the apparatus has been brought into an open system, for example,by taking off a stopper, etc., in oder to discard the incubation systemcontaining the pathogenic microorganisms, such a treatment is not onlytroublesome and risky but also requires professional technique and greatskill, even installations. Furthermore, since, in addition to thecontainer for incubation, it is always necessary to prepare adisinfectant solution for the treatment, it is troublesome in view ofportability and convenience in use.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an apparatus (ordevice) for detecting microorganisms whereby pathogenic microorganismssuch as those causing food poisoning and resistant microorganisms can bedetected and identified by simple operations and, after use, theapparatus can be safely and conveniently disposed.

Another object of the present invention is to provide an apparatus fordetecting microorganisms which can be not only used in hospitals butalso used privately or at home and which can be disposed by anyoneeasily and safely.

Still another object of the present invention is to provide an apparatusfor detecting microorganisms where its handling is simple whereby it canbe used at any time, previous or spontaneous countermeasure topathogenic microorganisms can be made very easy and disposal of theapparatus after use can be made easily and safely.

The present inventors have conducted an intensive study and found thatthe following constitution is very effective in achieving theabove-mentioned objects. A characteristic feature of a relativelyconvenient microorganism-detecting apparatus (wherein a medium which isto give an incubating condition suitable for the pathogenicmicroorganisms to be detected (such as specific microorganisms for foodpoisoning and microorganisms which are resistant to specificantibiotics) upon contacting the microorganisms, and amicroorganism-collecting part are located in a noncontact mannerrelative to each other and, when incubation for detecting themicroorganism is conducted, the specific medium andmicroorganism-collecting part are contacted with each other) is morefully utilized and, in addition, disposal (in some cases, disinfectingtreatment) of the apparatus after use can be conducted safely andeasily.

The apparatus for the detection of microorganisms in accordance with thepresent invention is based upon the above finding.

In one of its aspects, the present invention provides:

(1) an apparatus for detecting a microorganism, characterized in that,the apparatus comprises at least:

(a) a container for holding a medium which is used for culturing themicroorganism to be detected during the period of the incubation of themicroorganism,

(b) a part where the microorganism is collected,

(c) structure for allowing a person to hold the medium for culturing themicroorganism to be detected, in a noncontact manner with themicroorganism-collecting part (b), until the incubating stage of themicroorganism, and

(d) structure for disinfecting the medium after incubation;

(2) the apparatus for detecting the microorganism according to the above(1) wherein the structure (d) (structure for disinfecting the mediumafter incubation) includes structure for allowing a disinfectant to beheld, without contact with the microorganism, until the completion ofthe incubation for the microorganism to be detected;

(3) the apparatus for detecting the microorganism according to the above(1) or (2) wherein the medium and the disinfectant are made in such amanner that they can unitedly contact each other, by the action of aforce applied from outside, after incubation of the microorganism;

(4) the apparatus for detecting the microorganism according to any ofthe above (1) to (3) wherein the microorganism-collecting part (b) andthe medium used for culturing the microorganism to be detected are madein such a manner that they can unitedly contact each other, by theaction of a force applied from outside, during the incubation of themicroorganism;

(5) the apparatus for detecting the microorganism according to any ofthe above (1) to (4) wherein the apparatus contains;

(a) a hollow container having an opening at one of its ends,

(b) a microorganism-collecting part located in the hollow container,

(c) a medium for culturing the microorganism, the medium being locatedso as to contact the microorganism-collecting part during the incubationof the microorganism, and

(d) a disinfectant being located so as to contact the medium forculturing the microorganism after the incubation of the microorganism;

(6) the apparatus for detecting the microorganism according to any ofthe above (1) to (5) wherein the antibiotic substance is added to themedium;

(7) the apparatus for detecting the microorganism according to any ofthe above (1) to (6) wherein an antibiotic substance is contained, atleast part of the antibiotic substance coming unitedly into contact withthe medium, by the action of an external force, during the incubation ofthe microorganism, and at least the antibiotic substance and the mediumbeing located in the holding container in a noncontact manner;

(8) the apparatus for detecting the microorganism according to any ofthe above (1) to (7) wherein the microorganism is detected throughincubating the microorganism in a medium and observing the changes inthe medium, characterized in that the apparatus comprises

(i) a microorganism-collecting part,

(ii) an antibiotic substance,

(iii) a medium and

(iv) a disinfectant,

wherein at least a part of (i) the microorganism-collecting part, (ii)the antibiotic substance and (iii) the medium can unitedly contact eachother, by the action of a force from outside, during the incubation ofthe microorganism, at least the above-mentioned antibiotic substance(ii) and the above-mentioned medium (iii) being located in a storagecontainer in a noncontact manner, and

wherein, after incubation of the microorganism, at least the incubatedmicroorganism and the disinfectant (iv) can unitedly contact each otherby the action of a force from outside, at least the above-mentioneddisinfectant (iv) and the above-mentioned medium (iii) being located inthe storage container in a noncontact manner;

(9) the apparatus for detecting the microorganism according to any ofthe above (1) to (5) and (7) to (8) wherein the antibiotic substance is

(1) located in the above-mentioned container,

(2) given to the above mentioned microorganism-collecting part or

(3) coated on the inner wall of the above-mentioned container;

(10) the apparatus for detecting microorganism according to any of theabove (1) to (5) and (7) to (9) wherein the antibiotic substance isgiven to other materials located in the hollow container;

(11) the apparatus for detecting the microorganism according to any ofthe above (1) to (5) and (7) to (9) wherein the microorganism-collectingpart has a multiple structure containing at least an inside material andan outside material and the antibiotic substance is located in theinside material;

(12) the apparatus for detecting the microorganism according to any ofthe above (1) to (11) wherein the medium contains a substance capable ofchanging its color according to the change caused by growth of themicroorganism;

(13) the apparatus for detecting the microorganism according to any ofthe above (1) to (12) wherein the container has a cover for tightclosing of the container and the medium is located in the cover in sucha manner that it is enclosed in a bag-shaped member or vessel;

(14) the apparatus for detecting the microorganism according to theabove (13) wherein a perforated member is located between the bag-shapedmember and the hollow container;

(15) the apparatus for detecting the microorganism according to theabove (14) wherein a guide member which can promote a fall of the mediumdescending along the microorganism-collecting part is located under theperforated member;

(16) the apparatus for detecting the microorganism according to any ofthe above (1) to (15) wherein the apparatus has a incubation selectivityfor at least one microorganism selected from the group consisting ofpathogenic Escherichia coli, Staphylococcus aureus, Vibrioparahaemolyticus and Salmonella;

(17) the apparatus for detecting the microorganism according to any ofthe above (1) to (16) wherein the apparatus has a structure for allowinga person to disinfect the medium used for the incubation under a tightlyclosed state; and

(18) the apparatus for detecting the microorganism according to any ofthe above (1) to (17) wherein the apparatus has a structure for allowinga person to conduct at least

(i) addition of a medium into a container,

(ii) incubation,

(iii) visual detection of the microorganism cells, and

(iv) disinfecting treatment of the incubated medium under a tightlyclosed state after collecting of the microorganism by themicroorgansim-collecting part.

The present invention further contemplates:

(19) an apparatus for detecting a microorganism, characterized in that,the apparatus consists of (i) a container having a space wherein amedium is received and the microorganism is cultured and (ii) a coverwhich engages with an opening of the container to tightly close (fastenor seal) the opening of the container from the surrounding atmosphere(outside),

the cover being a receptacle body having (A) a first bag-shaped memberor vessel for holding (enclosing) the medium and (B) a second bag-shapedmember or vessel for holding (enclosing) a disinfectant;

(a) the receptacle body being bestowed with a function for breaking theinner bag-shaped member by the action of a force applied from outside inorder to remove the contents from the first bag-shaped member,

(b) the second bag-shaped member capable of being broken independentlyof the first bag-shaped member whereby the contents in the secondbag-shaped member are removed therefrom;

(c) at or near a connecting portion of the cover engaged with theopening of the container, a partition being formed or a partition memberbeing disposed for keeping the bag-shaped members in the cover;

(d) the partition or the partition member being equipped with amicroogranism-collecting part which is positioned in a protruding mannerat the side of a space of the container for receiving the medium andculturing the microorganism therein;

(e) wherein the end of the microorganism-collecting part can contact themedium in such manner that it is possible to culture the microorganismwhen the container receives the medium; and

(f) wherein one or more through holes are formed in the partition or thepartition member so that the medium and the disinfectant can flowbetween the cover and the container;

(20) the apparatus for detecting the microorganism according to theabove (19), characterized in that,

each of the first and the second bag-shaped members is a glass ampule,

the cover is made of a flexible material and,

even when a force from outside (an external force) is applied to thecover for breaking the first bag-shaped member to remove the mediumwhich is a content therein, the second bag-shaped member is not damagedand the disinfectant which is a content therein can be kept as stored;

(21) the apparatus for detecting the microorganism according to theabove (19), characterized in that,

the cover is composed of a first section for receiving the firstbag-shaped member and a second section for receiving the secondbag-shaped member,

the first section being installed with a first pushing-down member forbreaking the first bag-shaped member and

the second section being installed with a second pushing-down member forbreaking the second bag-shaped member.

(22) the apparatus for detecting the microorganism having a structure asshown in any of FIGS. 6 to 11 or characterized in having substantiallythe same function concerning the bactericidal treatment of the incubatedmicroorganism culture;

(23) An apparatus for detecting the microorganism according to the above(19), characterized in that

the cover is composed of:

(a) a first cover-constituting member which constitutes a first sectionfor receiving the first bag-shaped member,

(b) a second cover-constituting member which constitutes a secondsection for receiving the second bag-shaped member, and

(c) a cap member being located at one end of the secondcover-constituting member and at the opposite side of the fitting side(of the second cover-constituting member) for the firstcover-constituting member and slidably fitted into the secondcover-constituting member,

wherein, at or near each end of the first and second cover-constitutingmembers, each end being at the side of the container having a space forreceiving the medium to culture the microorganism therein, a partitionis formed or a member for the partition is disposed for preventing eachof the bag-shaped members from entering the container;

the first cover-constituting member being slidably engaged with one endof the second cover-constituting member wherein the first bag-shapedmember can be broken to release the medium contained therein by slidingthe second cover-constituting member to the side of the firstcover-continuing member by a force from outside (external force),

wherein the second bag-shaped member can be broken to release adisinfectant contained therein by sliding the cap member to the side ofthe second cover-constituting member by a force from outside (externalforce), and

wherein one or more through holes are formed in the partition or thepartition member in a manner that the medium and the disinfectant can becommunicated with the container;

(24) the apparatus for detecting the microorganism according to theabove (23), characterized in that,

an engagement of the first cover-constituting member with one end of thesecond cover-constituting member is achieved by use of a screw threadand a force is generated by rotating the second cover-constitutingmember against the first cover-constituting member by a force fromoutside (external force) whereby the first bag-shaped member can bebroken to release the medium which is a content therein;

(25) the apparatus for detecting the microorganism according to theabove (23), characterized in that,

an engagement of the second cover-constituting member with the capmember is achieved by use of a screw thread and an external force isgenerated by rotating the cap member against the secondcover-constituting member whereby the second bag-shaped member can bebroken to release the disinfectant which is a content therein;

(26) the apparatus for detecting the microorganism according to theabove (24), characterized in that,

the first and second cover-constituting members are constituted from acylindrical body;

(27) the apparatus for detecting the microorganism having an arrangementas shown in FIG. 12 or FIG. 13 or characterized in having substantiallythe same function concerning the bactericidal treatment of the culturedmicroorganism;

(28) the apparatus for detecting the microorganism according to theabove (19), characterized in that,

the cover is composed of:

(a) a first cover-constituting member which has a first section housinga first bag-shaped member and a second section receiving a secondcover-constituting member housing a second bag-shaped member,

(b) the second cover-constituting member and

(c) a cap member which is slidably engaged with one end of the secondcover-constituting member and is located thereon at the opposite side ofthe container having a space for receiving the medium to culture themicroorganism therein;

wherein, at or near each end of the first and second cover-constitutingmembers, each end being at the side of the container having a space forreceiving the medium to culture the microorganism therein, a partitionis formed or a member for the partition is disposed for preventing eachof the bag-shaped members from entering the container;

the first cover-constituting member being slidably engaged with one endof the second cover-constituting member wherein the first bag-shapedmember can be broken to release the medium contained therein by slidingthe second cover-constituting member to the side of the firstcover-constituting member by a force from outside (external force),

wherein the second bag-shaped member can be broken to release adisinfectant contained therein by sliding the cap member to the side ofthe second cover-constituting member by a force from outside (externalforce), and

wherein one or more through holes are formed in the partition or thepartition member in a manner that the medium and the disinfectant can becommunicated with the container;

(29) the apparatus for detecting the microorganism according to theabove (28), characterized in that,

an engagement of the first cover-constituting member with a portion ofthe second cover-constituting member is achieved by use of a screwthread and a force is generated by rotating the secondcover-constituting member against the first cover-constituting member bya force from outside (external force) whereby the first bag-shapedmember can be broken to release the medium which is a content therein;

(30) the apparatus for detecting the microorganism according to theabove (28), characterized in that,

an engagement of the second cover-constituting member with the capmember is achieved by use of a screw thread and an external force isgenerated by rotating the cap member against the secondcover-constituting member whereby the second bag-shaped member can bebroken to release the disinfectant which is a content therein;

(31) the apparatus for detecting the microorganism according to theabove (28), characterized in that,

the first and second cover-constituting members are constituted from acylindrical body;

(32) the apparatus for detecting the microorganism according to theabove (28), characterized in that;

most of the second cover-constituting member is housed in the firstcover-constituting member;

(33) the apparatus for detecting the microorganism as shown in FIG. 14or characterized in having a function substantially equivalent theretoconcerning the bactericidal treatment of the incubated microorganism;

(34) the apparatus for detecting the microorganism according to theabove (19), characterized in that,

the cover is composed of:

(a) a receptacle body receiving both of a first bag-shaped member and asecond bag-shaped member in a parallel and

(b) two tools for breaking the bag-shaped members, the tools beingslidably engaged with the top of the receptacle body at the oppositeside of the container having a space for receiving the medium to culturethe microorganism,

wherein the tools are independently pushed into the receptacle body bysliding via a force from outside where, first, the first tool can breakthe first bag-shaped member to release the medium contained therein andthen the second tool can break the second bag-shaped member to releasethe disinfectant contained therein, and

wherein through holes are made in the partition or the partition memberso that the medium and the disinfectant can be communicated with thecontainer;

(35) the apparatus for detecting the microorganism according to theabove (34), characterized in that,

the tools are disposed in such a manner that they can be pushed into thetop of the receptacle body via a force from outside;

(36) the apparatus for detecting the microorganism according to theabove (34), characterized in that,

the tools are disposed in such a manner that they can be pushed into thetop of the receptacle body in a screwing manner.

(37) the apparatus for detecting the microorganism according to theabove (34), characterized in that,

the outer appearance of the receptacle body is of a structure similar toa rectangular parallelepiped.

(38) the apparatus for detecting the microorganism as shown in FIG. 15or characterized in having a function substantially equivalent theretoconcerning the bactericidal treatment of the incubated microorganism;

(39) the apparatus for detecting the microorganism according to theabove (19), characterized in that

the cover consists of:

a receptacle body receiving a first bag-shaped member and a secondbag-shaped member in parallel,

the receptacle body consisting of a holding member which constitutes aholding section capable of regulating the position of the bag-shapedmembers and a movable material which constitutes a movable section beingrotatable by a force from outside against the holding section,

wherein a tool for breaking the bag-shaped member is installed on theinner side of the receptacle body,

wherein, when the movable member is rotated, any of the first bag-shapedmember and said second bag-shaped member can be independently broken bythe tool, and

wherein through holes are made in the partition or the partition memberso that the medium and the disinfectant can be communicated with thecontainer;

(40) the apparatus for detecting the microorganism according to theabove (39), characterized in that,

a tool is installed on the inner side of the movable member in thereceptacle body;

(41) the apparatus for detecting the microorganism according to theabove (39), characterized in that,

the receptacle body is of a cylindrical shape and,

when the movable member of the receptacle body is rotated, any of thefirst bag-shaped member and the second bag-shaped member can beindependently broken depending upon the direction of rotation; and

(42) the apparatus for detecting the microorganism as shown in FIG. 16or characterized in having substantially the same function concerningthe bactericidal treatment of the incubated microorganism.

The present invention further provides:

(43) an apparatus for detecting a microorganism, characterized in thatthe apparatus comprises:

(a) a hollow container in a cylindrical shape for holding a medium usedfor culturing the microorganism to be detected during the incubation ofthe microorganism,

(b) a microorganism-collecting part comprising a rod-like material and amicroorganism-collecting end and

(c) a cover equipped with the microorganism-collecting part wherein thecover stores at least two bag-shaped members where the first bag-shapedmember is to receive the medium while the second one is to receive adisinfectant (bactericide), and

wherein (d) the medium enclosed in the first bag-shaped member cancontact the microorganism-collecting end without detaching the coverwhich closes the opening of the hollow container (a) and thedisinfectant/bactericide enclosed in the second bag-shaped member cancontact the microorganism in the hollow container to perform adisinfecting/bactericidal treatment;

(44) the apparatus for detecting the microorganism according to theabove (43), characterized in that,

the cover is constituted from a material which can be easily deformed bya force from outside and the first and second bag-shaped members can beeasily broken by a force from outside to discharge the liquid contentsthere of;

(45) the apparatus for detecting the microorganism according to theabove (43), characterized in that,

the cover has a partition member at the side of the connecting part withthe hollow container and the partition member has an action of receivingand holding the first and the second bag-shaped members in the cover;

(46) the apparatus for detecting the microorganism according to theabove (43), characterized in that,

the partition member is capable of engaging with the cover at the sideof the part (of the cover) connecting with the hollow container; and

(47) the apparatus for detecting the microorganism according to theabove (43), characterized in that,

the cover is constituted in such a manner that each of the first andsecond bag-shaped members is independently broken by a force fromoutside so that the liquid contents thereof can be discharged.

The present invention further provides:

(48) the apparatus for detecting the microorganism according to theabove (19), characterized in that,

the cover is composed of:

(a) a first cover-constituting member which constitutes a first sectionreceiving and holding a first bag-shaped member,

(b) a second cover-constituting member which constitutes a secondsection receiving and holding a second bag-shaped member and

(c) a cap member which is rotatably screw-fitted at one end of thesecond cover-constituting member, the cap member being located at theside thereof opposite to the connected side for the firstcover-constituting member,

wherein either a partition is formed or a member for the partition isdisposed for keeping each of the bag-shaped members at or neat the endof the container, of the first and the second cover-constitutingmembers, having a space for receiving the medium to culture themicroorganism therein,

wherein the first cover-constituting member is slidably fixed with oneend of the second cover-constituting member and the secondcover-constituting member can be slid to the side of the firstcover-constituting member by a force from outside so that the firstbag-shaped member would be broken to release the medium containedtherein,

wherein the cap member can be rotated by a force from outside to pushinto the side of the second cover-constituting member so that the secondbag-shaped member would be broken to release a disinfectant containedtherein, and

wherein through holes are formed in the partition or the partitionmember so that the medium and the disinfectant can be communicated withthe space;

(49) the apparatus for detecting the microorganism according to theabove (48), characterized in that,

an engagement of the first cover-constituting member with one end of thesecond cover-constituting member is secured by use of an uneven surface(e.g. an indented surface including a recess and a protruding portion)made on the contacting surfaces between the members and, when the bodyof the second cover-constituting member is slid by a force from outsideto the side of the cover-constituting member, the first bag-shapedmember can be broken whereby the medium contained therein can beremoved.

(50) the apparatus for detecting the microorganism according to theabove (48), characterized in that,

an engagement of the second cover-constituting member with the capmember is in a screw-fixed manner and, the cap member can be rotatedrelative to the second cover-constituting member by a force from outsideand pushed into a side of the container, whereby the second bag-shapedmember would be broken to discharge the disinfectant contained therein;

(51) the apparatus for detecting the microorganism according to theabove (48), characterized in that,

the first and second cover-constituting members are constituted from acylindrical body;

(52) the apparatus for detecting the microorganism as shown in FIG. 17or FIG. 18 or that which is characterized in having substantially thesame function concerning the bactericidal treatment of the incubatedmicroorganism;

(53) the apparatus for detecting the microorganism according to theabove (52), equipped with a cap member having the structure as shown inFIG. 19 or FIG. 20 or that which is characterized in havingsubstantially the same function;

(54) the apparatus for detecting the microorganism according to theabove (52), equipped with a cover-constituting member having thestructure as shown in FIG. 22 or FIG. 23 or that which is characterizedin having substantially the same function;

(55) the apparatus for detecting the microorganism according to theabove (52), equipped with a cover-constituting member having thestructure as shown in FIG. 26 or FIG. 27 or that which is characterizedin having substantially the same function;

(56) the apparatus for detecting the microorganism according to theabove (48), characterized in that,

one or more convex parts on the side of the first cover-constitutingmember are formed as a structure having different installing regions,such as 781, 782, and

one or more concave parts suitably engaged with the convex part(s) areformed on the contacting surface of the second cover-constitutingmembers, the contacting surface contacting the first cover-constitutingmember,

wherein, before use, it is possible to engage the firstcover-constituting member with the second cover-constituting member insuch a manner that the concave part is not inserted into the convex partwhereby, the second cover-constituting member would be fixed within thefirst cover-constituting member in a nonsliding manner, and, upon use,it is possible to move the convex part into the concave part by rotatingthe second cover-constituting member, to ensure an engagement of thefirst cover-constituting member with one end of the secondcover-constituting member by use of the concavities and convexities madeon the contacting surfaces thereof and, to slide the body of the secondcover-constituting member to the side of the first cover-constitutingmember via a force from outside whereby the first bag-shaped memberwould be broken and the medium contained therein removed;

(57) the apparatus for detecting the microorganism according to theabove (56), characterized in being equipped with a cover-constitutingmember having the structure as shown in FIGS. 36 to 40 or that which hassubstantially the same function; and

(58) the apparatus for detecting the microorganism according to theabove (56), characterized in being equipped with a cover-constitutingmember having the structure as shown in FIGS. 41 to 44 or that which hassubstantially the same function.

Still another aspect of the present invention is to provide:

(59) the apparatus for detecting the microorganism according to any ofthe above (1) to (58), comprising a medium selected from the groupconsisting of:

(a) a medium for Salmonella having a composition substantiallycontaining an appropriate amount, such as 3 to 7 g, of tryptone, anappropriate amount, such as 1 to 6 g, of yeast extract, an appropriateamount, such as 5 to 15 g, of lysine, an appropriate amount, such as 0.5to 2 g, of glucose, an appropriate amount, such as 7 to 9 g, of sodiumchloride, an appropriate amount, such as 1.0 to 2.0 9, of monopotassiumdihydrogen phosphate, an appropriate amount, such as 0.1 to 0.3 g, ofsodium thiosulfate, an appropriate amount, such as 0.2 to 0.4 g, ofammonium iron citrate, an appropriate amount, such as 15 to 25 g, ofmagnesium chloride, an appropriate amount, such as 27 to 33 ml, of 0.4%Malachite Green solution and an appropriate amount, such as 0.01 to 0.03g, of Bromcresol Purple per 1,000 ml of the medium (pH of the mediumbeing about 5.3 to 5.7);

(b) a medium for Vibrio parahaemolyticus having a compositionsubstantially containing an appropriate amount, such as 25 to 40 g, of asalt polymyxin broth (the salt polymyxin broth contains an appropriateamount of yeast extract, an appropriate amount of peptone, anappropriate amount of sodium chloride and an appropriate amountpolymyxin B), an appropriate amount, such as 15 to 25 g, of mannitol, anappropriate amount, such as 5 to 10 g, of sodium citrate, an appropriateamount, such as 0.1 to 0.3 g, of sodium thiosulfate and an appropriateamount, such as 0.01 to 0.03 g, of Bromocresol Purple per 1,000 ml ofthe medium (pH of the medium being about 7.0 to 7.4);

(c) a medium for Escherichia coli group having a compositionsubstantially containing an appropriate amount, such as 26 to 43 g, of alauryl sulfate broth (the lauryl sulfate broth contains an appropriateamount of tryptose, an appropriate amount of lactose, an appropriateamount of monopotassium dihydrogen phosphate, an appropriate amount ofdipotassium monohydrogen phosphate, an appropriate amount of sodiumchloride and an appropriate amount of sodium lauryl sulfate), anappropriate amount, such as 3 to 8 g, of lactose and an appropriateamount, such as 0.03 to 0.05 g, of Bromthymol Blue or Bromocresol Purpleper 1,000 ml of the medium (pH of the medium being about 6.75 to 7.25);and

(d) a medium for Staphylococcus having a composition substantiallycontaining an appropriate amount, such as 5 to 15 g, of tryptone, anappropriate amount, such as 2 to 8 g, of yeast extract, an appropriateamount, such as 5 to 15 g, of mannitol, an appropriate amount, such as 2to 8 g, of dipotassium monohydrogen phosphate, an appropriate amount,such as 5 to 6 g, of lithium chloride, an appropriate amount, such as 12to 20 g, of glycine, an appropriate amount, such as 10 to 14 g, ofsodium pyruvate, an appropriate amount, such as 12 to 18 ml, of 1%aqueous potassium tellurite solution and an appropriate amount, such as0.02 to 0.03 g, of Phenol Red per 1,000 ml of the medium (pH of themedium being about 7.25 to 7.75);

(60) the apparatus for detecting the microorganism according to any ofthe above (48) to (58), characterized in using a medium selected fromthe improved media described in Example 11; and

(61) the apparatus for detecting the microorganism according to theabove (1), characterized in that,

after collecting the sample for incubation by use of themicroorganism-collecting part, all steps:

(a) supply of the medium into a container,

(b) incubation of the microorganism on the medium in the container,

(c) detection and/or finding of the microorganisms and

(d) disinfection and/or sterilization of the medium in the containerafter incubation can be conducted in a substantially tightly closedsystem and the apparatus is portable.

Another aspect of the present invention is to provide:

(62) the apparatus for detecting the microorganism according to any ofthe above (1) to (5) wherein the microorganism is detected throughincubating the microorganism in a medium and observing the changes inthe medium, characterized in that the apparatus comprises

(i) a microorganism-collecting part,

(ii) a medium and

(iii) a disinfectant,

wherein at least a part of (i) the microorganism-collecting part and(ii) the medium can unitedly contact each other, by the action of aforce from outside, during the incubation of the microorganism, theabove-mentioned microorganism-collecting part (i) and theabove-mentioned medium (ii) being at least located in a storagecontainer in a noncontact manner, and

wherein, after incubation of the microorganism, at least the incubatedmicroorganism and the disinfectant (iii) can unitedly contact each otherby the action of a force from outside, at least the above-mentioneddisinfectant (iii) and the above-mentioned medium (ii) being located inthe storage container in a noncontact manner;

(63) the apparatus for detecting the microorganism according to any ofthe above (1) to (62) wherein the container comprises a cover forclosing and opening, the cover accommodating a bag-shaped member whichencloses the medium;

(64) the apparatus for detecting the microorganism according to any ofthe above (1) to (63) wherein the container comprises a cover member forclosing and opening the container, the cover member accommodating abag-shaped member which encloses the disinfectant;

(65) the apparatus for detecting the microorganism according to any ofthe above (1) to (64) wherein the cover has a structure for allowing itto hold and carry at least

(i) a first bag-shaped member containing a medium, and

(ii) a second bag-shaped member containing a disinfectant independentlyeach other;

(66) the apparatus for detecting the microorganism according to any ofthe above (1) to (65) wherein the cover comprises at least

(i) a first cover-constituting member accommodating the first bag-shapedmember, and

(ii) a second cover-constituting member accommodating the secondbag-shaped member;

(67) the apparatus for detecting the microorganism according to any ofthe above (1) to (66), comprising

structure for protection and engagement, being disposed in the first andsecond cover-constituting members so as not to break the firstbag-shaped member by moving the second cover-constituting member priorto use;

(68) the apparatus for detecting the microorganism according to any ofthe above (1) to (67), wherein the second cover-constituting member isequipped with a cap member and structure for protection and engagementis disposed in the second cover-constituting member and the cap memberso as not to break the second bag-shaped member by moving the cap memberprior to use;

(69) the apparatus for detecting the microorganism according to theabove (67) or (68), wherein the structure for protection and engagementis a combination of a stopper (1005, 1008) and a guide (1004, 1007);

(70) the apparatus for detecting the microorganism according to any ofthe above (67) to (69), wherein the guide has a shape selected from thegroup consisting of shape a, b, c and d as shown in FIG. 70;

(71) the apparatus for detecting the microorganism having a structure asshown in any of FIGS. 53 to 64 or characterized in having substantiallythe same function concerning the bactericidal treatment of the incubatedmicroorganism culture;

(72) a method for quantitatively measuring the number of viablemicroorganism cells in a sample or specimen through using the apparatusfor detecting the microorganism according to any of the above (1) to(58) and (60) to (70);

(73) the method according to the above (72), wherein the measurement iscarried out in a closed system after collecting a microorganism to bemeasured and transferring it into the apparatus according to any of theabove (1) to (58) and (60) to (70); and

(74) the method according to the above (72) or (73), wherein a target isthe number of viable microorganism cells prior to incubation.

The above objects and other objects, features, and advantages of thepresent invention are readily apparent from the following detaileddescription of the best mode for carrying out the invention when takenin connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring to the several drawing figures, wherein like numerals refer tolike parts throughout, and wherein:

FIG. 1 is a schematic side cross sectional view showing an embodiment ofthe microorganism-detecting apparatus of the present invention;

FIGS. 2a and 2 b are schematic views showing an example of how to usethe microorganism-detecting apparatus of FIG. 1;

FIG. 3 is a schematic view showing an example of how to use themicroorganism-detecting apparatus of FIG. 1;

FIG. 4 is a schematic side cross sectional view showing anotherembodiment of the microorganism-detecting apparatus of the presentinvention;

FIG. 5 is a schematic side cross sectional view showing anotherembodiment of the microorganism-detecting apparatus of the presentinvention;

FIG. 6 shows another embodiment of the microorganism-detecting apparatusof the present invention where the cross-sectional shape of theapparatus is mainly illustrated;

FIG. 7 mainly shows the outer shape of the apparatus of FIG. 6;

FIG. 8 shows the outer shape of the container part supplying a space forincubation of a microorganism in the apparatus of FIG. 6;

FIGS. 9a and 9 b show the cross-sectional structure of the containerpart supplying the space for incubation of a microorganism in theapparatus of FIG. 6 wherein 9(a) is a shape from its top while 9(b) is ashape from its side;

FIGS. 10a, 10 b and 10 c show an outer shape of the cover part of theapparatus of FIG. 6 wherein 10(a) is a shape from its bottom, 10(b) is ashape from its side, and 10(c) is a shape from its top;

FIG. 11 shows a cross-sectional structure of the cover part of theapparatus of FIG. 6;

FIG. 12 shows another embodiment of the microorganism-detecting of thepresent invention where the outer shape is illustrated;

FIG. 13 shows another embodiment of the microorganism-detectingapparatus of the present invention where the cross-sectional shape ofthe apparatus of FIG. 12 is illustrated;

FIGS. 14a, 14 b and 14 c show another embodiment of themicroorganism-detecting apparatus of the present invention where 14(a)is a ring (77), 14(b) is a view from the side of themicroorganism-detecting apparatus consisting of container and cover, and14(c) is a cross-sectional shape of the cover part equipped with (73)and (74);

FIGS. 15a and 15 b show another embodiment of themicroorganism-detecting apparatus of the present invention where 15(a)is a shape from its top while 15(b) is a shape from its side;

FIGS. 16a, 16 b and 16 c show another embodiment of themicroorganism-detecting apparatus of the present invention where 16(a)is a cross-sectional shape of the part corresponding to thecover-constituting member (112), 16(b) is a cross-sectional shape of thepart corresponding to the cover-constituting member (102) and 16(c) is ashape from the side of the microorganism-detecting apparatus;

FIG. 17 shows another embodiment of the microorganism-detectingapparatus of the present invention where the outer shape is illustrated;

FIG. 18 shows a cross-sectional shape of the apparatus of FIG. 17;

FIG. 19 shows an outer shape of a cap member (561) of the apparatus ofFIG. 17;

FIG. 20 shows a cross-sectional shape of a cap member (561) of FIG. 19;

FIG. 21 shows a cross-sectional shape along I-I′ of the cross sectionalview of FIG. 20;

FIG. 22 shows an outer shape of the cover-constituting member (247) ofthe apparatus of FIG. 17;

FIG. 23 shows a cross-sectional shape of the cover-constituting member(247) of FIG. 22;

FIG. 24 shows an outer appearance of the cover-constituting member (247)of FIG. 22 from the direction of II;

FIG. 25 shows an outer appearance of the cover-constituting member (247)of FIG. 22 from the direction of III;

FIG. 26 shows an outer shape of the cover-constituting member (243) ofthe apparatus of FIG. 17;

FIG. 27 shows a cross-sectional shape of the cover-constituting member(243) of FIG. 26;

FIG. 28 shows an outer appearance of the cover-constituting member (243)of FIG. 26 from the direction of IV;

FIG. 29 shows an outer appearance of the cover-constituting member (243)of FIG. 26 from the direction of V;

FIG. 30 shows an outer shape of the container (241) of the apparatus ofFIG. 17;

FIG. 31 shows a cross-sectional shape of the container (241) of FIG. 30;

FIG. 32 shows an outer appearance of the container (241) of FIG. 30 fromthe direction of VI;

FIG. 33 shows a cross sectional shape along G-G′ in the cross section ofFIG. 31, wherein a cross section of the microorganism-collecting end(246 b) is illustrated at its central portion;

FIG. 34 is a schematic view of composition of the members whichconstitute the microorganism-detecting apparatus of the presentinvention;

FIG. 35 shows an embodiment of the microorganism-detecting apparatus ofthe present invention and a cross-sectional shape is shown here;

FIG. 36 shows an outer shape of the cover-constituting member (747) ofFIG. 35;

FIG. 37 is a cross-sectional view taken along lines M-M′ of thecover-constituting member (747) of FIG. 36;

FIG. 38 shows an outer shape of the cover-constituting member (747) ofFIG. 36 from the direction of VII;

FIG. 39 shows an outer shape of the cover-constituting member (747) ofFIG. 36 from the direction of VIII;

FIG. 40 shows a cross-sectional shape of the cover-constituting member(747) of FIG. 36;

FIG. 41 shows a cross-sectional shape of the cover-constituting member(743) of FIG. 35;

FIG. 42 shows an outer shape of the cover-constituting member (743) ofFIG. 41 from the direction of IX;

FIG. 43 shows an outer shape of the cover-constituting member (743) ofFIG. 41 from the direction of X;

FIG. 44 shows a cross-sectional shape of the cover-constituting member(743) of FIG. 41;

FIG. 45 is a cross-sectional view taken along lines N-N′ of thecover-constituting member (743) of FIG. 44;

FIG. 46 is a cross-sectional view taken along lines P-P′ of thecover-constituting member (743) of FIG. 44;

FIG. 47 is a cross-sectional view taken along lines Q-Q′ of thecover-constituting member (743) of FIG. 44;

FIG. 48 is an oblique outer view of the cover-constituting member (743)of FIG. 41 which is made of a transparent (or translucent) material; and

FIG. 49 is a conceptional view showing the relationship between thecover-constituting member (743) and that (747) shown in FIG. 35 byshowing a small convex ridge (781) and a shallow concave groove (777).This is a state before incubation is started.

FIG. 50 is a conceptional view showing the relationship between thecover-constituting member (743) and that (747) shown in FIG. 35 byshowing a small convex ridge (781) and a shallow concave groove (777).This shows an operation when incubation is started.

FIG. 51 is a conceptional view showing the relationship between thecover-constituting member (743) and that (747) shown in FIG. 35 byshowing a small convex ridge (781) and a shallow concave groove (777).This is a state when the cover-constituting member (747) is pushed untila position where an ampule (744) is broken.

FIG. 52 shows a partial cross sectional view structure of a connectingpart with a container in a cover-constituting member (e.g., 743).

FIG. 53 is a perspective view of an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 54 illustrates perspectively the assembling of the parts 1245 (and1561) and 1243 for the microorganism-detecting apparatus of FIG. 53.

FIG. 55 is a perspective view of an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 56 illustrates perspectively the assembling of the parts 1245 (and1561) and 1243 for the microorganism-detecting apparatus of FIG. 55.

FIG. 57 is a perspective view of an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 58 illustrates perspectively the assembling of the parts 1561 and1245 for the microorganism-detecting apparatus of FIG. 57.

FIG. 59 is a perspective view of an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 60 illustrates perspectively the assembling of the parts 1561 and1245 for the microorganism-detecting apparatus of FIG. 59.

FIG. 61 is a perspective view of an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 62 illustrates perspectively the assembling of the parts 1561, 1245and 1243 for the microorganism-detecting apparatus of FIG. 61.

FIG. 63 is a perspective view of an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 64 illustrates perspectively the assembling of the parts 1561, 1245and 1243 for the microorganism-detecting apparatus of FIG. 63.

FIGS. 65A and 65B are views of the members (1561, 1245) for anembodiment of the microorganism-detecting apparatus according to thepresent invention (65A: front view and 65B: side view).

FIG. 66 is a side view of the members (1561, 1245) for an embodiment ofthe microorganism-detecting apparatus according to the presentinvention.

FIGS. 67A and 67B show schematically the member 1243 capable of engagingwith the member (1245) as illustrated in FIG. 65 (67A: top view and 67B:front side view).

FIG. 68 is a schematic diagram for explaining the relationship between aguide portion (1004) and a stopper (1005) for an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 69 illustrates schematically the enlarged engagement of a guideportion (1004) with a stopper (1005) at the site B for an embodiment ofthe microorganism-detecting apparatus according to the presentinvention.

FIG. 70 illustrates various shapes for the guides (1004, 1007) for aprotecting and engaging structure in an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 71 is a graph showing the relationship between the number of viableStaphylococcus aureus cells and measured turbidity data when themicroorganism was cultured in an embodiment of themicroorganism-detecting apparatus according to the present invention.

FIG. 72 is a graph showing the relationship between the number of viableEscherichia coli cells and measured turbidity data when themicroorganism was cultured in an embodiment of themicroorganism-detecting apparatus according to the present invention.

Explanation of the symbols (numerals) used is as follows:

1, 21, 41, 61, 81, 101, 241 and 741: main body of container

2, 22, 42, 62, 82, 102 and 242: cover

3: medium

4, 24, 44, 64 (or 68), 84, 104, 244 and 744: first bag-shaped member orvessel

6, 26, 46, 66, 86, 106, 246 and 746: microorganism-collecting member(part)

6 a, 26 a, 46 a, 66 a, 86 a, 106 a, 246 a and 746 a: rod-shaped element(stem)

5 and 296: first partition member

15 and 235: second partition members

6 b, 26 b, 46 b, 66 b, 86 b, 106 b, 246 b and 746b:microorganism-collecting end

7: disk member impregnated with antibiotic substance

9: disinfectant/sterilizer

20: guide member

11, 59, 79, 89, 109, 259 and 776: connecting part of the container withthe cover

8, 28, 48, 68 (or 64), 88, 108, 248 and 748: second bag-shaped member orvessel

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to an apparatus for the detection ofmicroorganisms which is particularly portable or is able to be carriedeasily, the apparatus being capable of initiating the incubation of themicroorganism immediately at the place where the microorganism iscollected and/or being capable of starting in the incubation of themicroorganism without skillfulness. It is understood that the apparatusfor detecting microorganisms in accordance with the present invention isrelatively small in size, portable and, after incubation of themicroorganisms, able to conduct the detecting steps until the disposalof the used apparatus in a substantially tightly closed systemcontaining the incubated microorganism.

The term “substantially tightly closed system” used here means that,although it is possible to incubate, find or detect the microorganismand to disinfect and/or sterilize the incubated microorganism to adisposable extent, the cultured pathogenic microorganism does notdiffuse to outside or to the environment during the incubating periodand/or the incubated pathogenic microorganism does not come to theoutside or environment by diffusion until the completion of itsdisinfection and/or sterilization, or it does not pollute the outside orenvironment, and the term further means that such a function can beachieved.

In the microorganism-detecting apparatus of the present invention asmentioned above, the medium which is to give an incubating conditionsuitable for the incubation of the microorganism, and themicroorganism-collecting part are located in a noncontact mannerrelative to each other upon a non-using stage, and during the incubationfor the detection of the microorganism, the specific medium andmicroorganism-collecting part contact each other whereby the statussuitable for proliferation of the microorganism is achieved and, afteruse, the apparatus can be safely disinfected and/or sterilized.

In the present invention, the medium is further made in an appropriatestate where the specific species of the microorganism (food poisoningmicroorganisms such as Staphylococcus aureus) are proliferated but othermicroorganisms (i.e. those other than Staphylococcus aureus such asSalmonella, Escherichia coli, Vibrio parahaemolyticus and otherintestinal microorganisms) are substantially unable to grow. After thedetection, a treatment for disposal of the apparatus can be conducted bya simple operation and a safe disposal is made possible at all times.

Accordingly, when an appropriate incubating structure (such as a simpleincubator) and detecting structure (such as a pH indicator which detectsthe changes in pH due to growth of the microorganism) of themicroorganism are utilized, it is now possible to easily detect thespecific species of microorganisms (such as food poisoning ones) andanyone is able to easily dispose the apparatus after use. When anapparatus which is in such a status that it gives an incubatingcondition having a selectivity to specific species of microorganisms(for example, food poisoning ones such as Staphylococcus aureus) isused, it is now easy to selectively detect and identify (i.e. todiscriminate the type of the food poisoning microorganism) the specificspecies of the microorganisms (such as food poisoning ones) and todisinfect and sterilize the apparatus containing the pathogenicmicroorganisms without anxiety.

Examples of the pathogenic microorganism are Staphylococcus aureus,Vibrio parahaemolyticus, Salmonella (such as Salmonella typhi,Salmonella paratyphi A, Salmonella schottmuelleri, Salmonellaenteritidies, Salmonella thompson, Salmonella narashino, Salmonellapotsdam, Salmonella oranienburg and Salmonella senftenberg), Clostridiumbotulinum, Shigella (such as Shigella dysenteriae and Shigellaflexneri), Vibrio cholerae, Vibrio cholerae biotype eltor andEscherichia coli (such as enteropathogenic Escherichia coli representedby E. coli O-157).

When the apparatus is used for detection of food poisoning, theapparatus of the present invention can be preferably made in such amariner that it shows a selective incubating ability for at least onemicroorganism selected from the group consisting of Staphylococcusaureus, Vibrio parahaemolyticus and Salmonella, which occupy nearly all(about 99%) of the microorganisms causing food poisoning while thediscrimination among those types of microorganism may not be essential.This is because, when the presence of at least one of these three typesof microorganisms is confirmed, then it is possible to substantiallyremove all of the three types of microorganisms from the environment, byapplying a certain disinfectant as mentioned later, to the environment.When the food poisoning microorganism can be detected and identified ina manner as mentioned above (such as spontaneously), it is now possibleto prevent food poisoning easily and effectively by adopting a measuresuitable for removing the food poisoning microorganism (such as by adisinfecting method where cooking devices, etc. are cleaned with“Hibiden” (trade name: SUMITOMO PHARMACEUTICALS CO. LTD., Japan, for a0.2 to 0.5% solution of chlorohexidine) or with an invert soap followedby wiping and washing with water).

On the other hand, if it is necessary to identify by discriminating eachof Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella andpathogenic Escherichia coli O-157 as a microorganism causing the foodpoisoning for the sake of the countermeasure, it is also possible that aselective incubating ability is given to the microorganism-detectingapparatus used, or a selective identifying ability is given to thedetecting function.

If necessary, the apparatus for detecting microorganisms in accordancewith the present invention may be constituted in such a manner that aselectivity for incubation of the “big three” food poisoningmicroorganisms (i.e. Staphylococcus aureus, Vibrio parahaemolyticus andSalmonella; about 99% of the bacterial food poisoning is caused by anyof them) is given. It is also possible to give a selectivity forincubation of enterohaemorrhagic Escherichia coli (such as E. coliO-157:H7) which has been an increasingly big problem in recent years. Itis further possible to give a selectivity for incubation ofantibiotic-resistant microorganism such as MRSA.

The present invention will now be further illustrated by, if necessary;referring to the attached drawings.

FIG. 1 and FIGS. 3 to 16 show outer appearances, cross sections alongthe side of the apparatus, etc. of one of the preferred embodiments ofthe present invention.

First, an illustration will be made by referring to FIG. 1. Thedetecting apparatus in this embodiment is constituted from a hollow andcylindrical container (1) having an opening at the upper end and a cover(2) which can be freely inserted into and detached from the opening. Atthe upper part of the cover (2) (i.e. an opposite side to the containeropening of the cover (2)), a first bag-shaped member or vessel is placedwhich encloses a medium (3) suitable for the incubation of a specificmicroorganism such as Staphylococcus aureus. At the upper part thereof,a second bag-shaped member or vessel (8) is placed which encloses adisinfectant (9) having a sufficient concentration or amount fordisinfecting and/or sterilizing the incubated microorganism. The medium(3) further contains a pH indicator (such as Phenol Red, BromcresolPurple and Bromthylmol Blue) which identifies the pH changes due toproliferation of the microorganism. On the other hand, at the lower side(an opening side of the container) of the first bag-shaped member (4), apartition member (5) having one or more (preferably, more than one)hole(s) is disposed (e.g. embedded) which prevents falling of the firstbag-shaped member (4) into an opening of the container. Further, at thelower side (at the side of the first bag-shaped member) of the secondbag-shaped member or vessel (4), a partition member (15) having one ormore (preferably, more than one) hole(s) is disposed (e.g. embeded)which prevents falling of the second bag-shaped member (8) downward. Inorder to restrict such a downward movement of the first bag-shapedmember (4) and the second one (8), there will be no need of using thepartition members (5 and 15) but the structure having the same function,including, for example, an element constituting the cover (2) and havingone or more through holes (i.e., a perforated portion in the partitionpart thereof), will suffice.

Further, at the nearly central part of the bottom side of the cover (2),a microorganism-collecting member (part) (6) extending to an almostvertical direction to the opening side of the above container is placed.The microorganism-collecting part (6) consists of a rod-like material(stem) (6 a) located at the side near the cover (2) and amicroorganism-collecting end (6 b) located at the end (a far side fromthe cover (2)) of the rod-like material. Usually, themicroorganism-collecting end (6 b) is received in such a manner that itdoes not contact with the medium before use. Themicroorganism-collecting end (6 b) is to achieve a selective incubationand, usually, the end is received in such a manner that it does notcontact with an antibiotic substance having a function of inhibiting thespecific microorganism. However, as will be mentioned below in detail,it may be, if necessary, in such a state that some contact with theantibiotic substance is available or that the antibiotic substance isgiven at the microorganism-collecting end.

Examples of the antibiotic substance are those having a characteristicnature of effectively inhibiting the proliferation of the microorganismsother than specific ones to be detected (e.g., food poisoningmicroorganisms such as enteropathogenic Escherichia coli and Salmonellaand resistant microorganisms such as MRSA). When microorganisms whichcause food poisoning (such as Staphylococcus aureus, Vibrioparahaemolyticus and Salmonella) are used as the microorganisms to bedetected, examples of the antibiotic substance are aztreonam, polymyxinB, fluconazole and a combination thereof. When an antibiotic-resistantmicroorganism is used as the microorganism to be detected, the examplesare oxacillin which inhibits Gram-positive bacteria having noresistance, polymyxin B and aztreonam which inhibits Gram-negativebacteria, polymyxin B as well as a combination thereof.

The antibiotic substance may, as will be mentioned later, be previouslyadded to the medium, coated on the part of the container (1) used forincubation (such as a bottom or the container (1) or a wall near there),present as a powder which is made to be easily dissolved, or coated atthe place which is a part of the microorganism-collecting part (such asthe place which is just above the microorganism-collecting end (6 b)).It is also possible that the antibiotic substance is placed separatelyfrom the part used for incubation in the above-mentioned container, themedium (3) or the microorganism-collecting part (6). In an embodimentwhere the antibiotic substance is placed separately as such, adisk-shaped member (7) (refer to FIG. 5; preferably composed of “porousmaterial”) to which the antibiotic substance is given may, if necessary,be placed at the inner area of the container (1) (such as a spacebetween the microorganism-collecting end (6 b) and the bottom of thecontainer). Alternatively, the antibiotic substance may be placed insuch a manner that it is supplied when the medium (3) enclosed in thefirst bag-shaped member (4) (which will be mentioned below) falls downinto the container. In such an embodiment, it is also possible that theantibiotic substance is placed at the partition member (5) or at theupper side of the guiding material (20) (cf. FIG. 4).

When a microorganism-detecting apparatus in such an embodiment as shownin FIG. 1 is used, a cover (2) having a microorganism-collecting part(6) is at first taken out from the container as shown in FIG. 2(a).Then, as shown in FIG. 2(b), the microorganism-collecting end (6 b) atthe end of the microorganism-collecting part (6) is rubbed on an objectto be tested (10) (such as a chopping board) and the microorganismexisting on the object to be tested (10) is collected on themicroorganism-collecting end (6 b). Then the cover (2) having themicroorganism-collecting part (6) is inserted into the container againand, after that, as shown in FIG. 3, the first bag-shaped member (4) isbroken (by, for example, strongly compressing it from outside of thecover (2) using a compressing structure which is not shown here) so thatthe medium (3) which is enclosed and received in said first bag-shapedmember (4) falls down into the container through the holes of thepartition member (5). As a result, the microorganism-collecting end (6b) of the microorganism-collecting part (6) is dipped into the medium(3). At that time, falling of the materials other than the medium suchas the broken pieces of the first bag-shaped member (4) is inhibited bythe partition member (5) and does not fall down into the container.

For example, as mentioned hereinabove, when an antibiotic substancehaving a specific selectivity is attached to themicroorganism-collecting end (6 b), proliferation of the microorganismsother than the specific microorganism (such as food poisoningmicroorganisms including Staphylococcus aureus and Clostridiumbotulinum) is substantially inhibited in case themicroorganism-collecting end (6 b) of the microorganism-collecting part(6) is dipped in the medium (3) as such. Accordingly, when themicroorganism-detecting apparatus in such a state is subjected to anincubation by an appropriate incubating structure (such as a simpleincubator), the specific microorganism (such as food poisoningmicroorganism including Staphylococcus aureus and Clostridium botulinum)is selectively proliferated. There is no particular limitation for theincubating condition at that time but, usually, the condition of 37° C.for about 16 to 24 hours is sufficient. After the above-mentionedincubating operation, a change, if any, of the pH indicator contained inthe medium (3) (and also the degree of changes in color) is checkedwhereby the specific food poisoning microorganism (such asStaphylococcus aureus) can be easily detected and identified (i.e.discrimination of the type of the food poisoning microorganism isconducted). For example, the medium containing Phenol Red as a pHindicator shows a pink color (pH: 7.4) in the initial stage ofincubation and, when Staphylococcus aureus is present, it changes toyellow whereby a detection can be done. In addition, physical andchemical changes in the nature and appearance resulting from thesubstances which are produced by the proliferated microorganism orobtained due to a decomposition thereby, turbidity of the medium andgrowth of colony are observed as a change in the medium whereby thepresence of the microorganism may be judged as well. From a viewpoint ofobservation by the naked eye as such, it is preferred that the container(1) has a light-transmitting ability such as that at least the areawherefrom the observation is conducted is transparent. When there is apossibility that the medium, the antibiotic substance, etc. are affectedby light to change their nature, then the transparent area may be insuch a constitution that it is covered by a light-shielding coat and,upon observation, it is detached.

Finally, the apparatus where the microorganism is incubated and detectedis disposed of and, for such a purpose, it is necessary that theincubated cells, cultured products, inner side of the container, etc.are completely disinfected/sterilized. Although not shown in thefigures, the second bag-shaped member (8) is broken (by applying asufficient force for disintegration or by strongly compressing fromoutside of the cover (2) using, for example, a disintegrating mechanismor compressing device which is not shown) whereby the disinfectant (9)enclosed and received in the second bag-shaped member (8) falls into thecontainer through the holes of the partitions (5 and 15). As a result,the microorganism-collecting end (6 b) of the microorganism-collectingpart (6) and the medium (3) containing the incubated microorganism aredipped in and/or mixed with the disinfectant (9). For a completedisinfection/sterilization at that time, it is preferred to shake thecontainer so that the disinfectant (9) comes throughout the inside ofthe container (1) and, since a treatment is conducted in a substantiallytightly-closed system in the present invention, it is possible toconduct a safe and sure disinfecting/sterilizing operation.

Constitution of each part of the apparatus for detection ofmicroorganisms in accordance with the present invention will beillustrated as follows:

Container

It is preferred that the container is constituted from a transparent ortranslucent material. To be more specific, the container may be formedusing an inorganic material such as glass or an organic material such asplastics (e.g., polyethylene, polypropylene, polystyrene, polyethyleneterephthalate (PET), polycarbonate, acryl resin and polyolefin) and,when weight and possibility of breakage upon centrifugal operation, etc.are taken into consideration, it is preferred that the container isconstituted from plastics.

There is no particular limitation for the shape of the container so faras it has an opening at the upper end and a cover (2) can be freelyattached to and detached from the opening. To be more specific, inaddition to the shape of a test tube as shown in the figures, any ofshapes in flask, bottle, etc. may be used.

Cover

There is no particular limitation for the material which constitutes thecover (2) but the material may be the same one as that used for thecontainer. In another embodiment, the cover (2) may be preferablyconstituted from a material which is softer than that used for thecontainer for lowering the possibility of damage of the container andfor increasing the contact adherence with said container. To be morespecific, both container and cover may be constituted from polypropylene(PP) or, when the container is constituted from hard plastics or hardresin such as polystyrene and PET, the cover (2) may be constituted fromsoft plastics such as polyolefin or soft resin (this term is intended touse for covering elastic materials or elastomers as well) or, in somecases, that is rather preferred. The cover is a material having a partplaced for connecting to a mouth or a wall of the container. The covermay be in such a form that it is fixed to the container by use of eithera screw thread or bayonet, or is fixed at the certain position bydeformation of a connecting part. It is also possible to place a packingor the like at the connecting part of the cover with the container. Allor a part of the material which constitutes the cover may be colored fordiscrimination or may be printed with letters, signs, pictures, etc.

In preferred specific examples, the cap part at the top of the cover maybe of a different color for each microorganism to be tested so that thetype, etc. of the microorganism to be tested can be easily understoodand the part may be suitably colored or modified with letters, signs,pictures, etc. It is also possible to use a material which has its colorby itself.

In the apparatus of the present invention, it is preferred that thecover (2) can receive at least two bag-shaped members as shown in FIG.1, FIGS. 4 to 7 and FIGS. 10 to 16, where the first bag-shaped memberreceives a medium while the second one receives adisinfectant/bactericide.

Bag-Shaped Material

A. Medium

There is no particular limitation for the material, shape, etc. of thefirst bag-shaped member or vessel (4) so long as the first bag-shapedmember (4) can surely and stably enclose the medium (3) upon storage andtransportation of the microorganism-detecting apparatus, and apredetermined amount of the medium (3) can be surely released uponincubation of the microorganism.

To be more specific, material for the first bag-shaped member (4) may bea hard material such as glass and hard plastics or a soft one such assoft plastics (e.g., polyethylene and polypropylene) and flexiblematerial (e.g., paper) but, in view of ease of confirming the presenceof the enclosed medium and the color tone, etc. thereof, it is preferredto use a transparent or translucent material.

In the present invention, there is no particular limitation for thestructure for releasing the medium (3) from the fist bag-shaped member(4) so long as the medium (3) is placed at a position appropriate forincubation upon the incubation of the microorganism. To be morespecific, upon releasing the medium (3) from the first bag-shaped member(4), the member (4) may be subjected to any of the operations includingperforation, cutting and breakage. When perforation, cutting, etc. ofthe first bag-shaped member (4) is conducted, a tool such as aneedle-like member or an edge-like member whereby perforation or cuttingis made easy (not shown in figures), may be installed at the inner wallof the cover (2) if necessary. On the other hand, in an embodiment wherethe medium (3) is released by breaking the first bag-shaped member (4),it is preferred in terms of ease of said breakage that the firstbag-shaped member (4) be composed of a hard material such as glass andhard plastics. In a structure where a substantially tight-sealed systemcan be maintained and a sufficient breaking force from outside can beapplied to the bag-shaped member, it is possible to easily apply astrong tension to such a hard material and to break it whereby thecontents can be removed therefrom. An example of the preferred structureis that a strong tension is concentrated on a specific area (that may betwo or more areas but a considerably narrow area is preferred) of thebag-shaped member made of a hard material. As will be mentioned indetail by referring to FIGS. 6 to 16 hereunder, it is to be understoodthat such breakage can be achieved by various structures, and it is alsopossible to adopt a structure where known structures and conventionalstructures are applied to the microorganism-detecting apparatus, orintalled within the microorganism-detecting apparatus of the presentinvention.

B. Disinfectant/Bactericide

In the present invention, there is no particular limitation for thematerial, shape, etc. of the second bag-shaped member or vessel (8) solong as the disinfectant/bactericide (9) can be safely and surelyenclosed and received by the second bag-shaped member (8) during thestorage/transportation of the microorganism-detecting apparatus untiluse, and during incubation of the microoganism, and so long as apredetermined amount of the disinfectant/bactericide (9) can be surelyreleased upon disposal of the apparatus after use.

To be more specific, the material of the bag-shaped member can beselected from those which are used for the already-mentioned firstbag-shaped member, and it is preferably a transparent or translucentmaterial with the purposes that the receiving disinfectant/bactericide(9) can be stably and surely enclosed and the presence of thepredetermined amount of the disinfectant/bactericide can be surelyconfirmed when the incubated microorganism is sterilized/disinfected, orthat confirmation of color tone, etc. can be done in any easy mannerfrom a viewpoint of confirmation that the sterilizing/disinfectingtreatment was surely done.

In the present invention, there is no particular limitation for thestructure for releasing the disinfectant/bactericide (9) from the secondbag-shaped member so long as the disinfectant/bactericide is positionedin a place suitable for the disinfecting/sterilizing treatment upon thedisposing stage of the microorganism-detecting apparatus. To be morespecific, it is possible to use any structure capable perforating,cutting, breaking, etc. the second bag-shaped member (8) for releasingthe disinfectant/bactericide (9) from the second bag-shaped member (8).When perforation, cutting, etc. of the second bag-shaped member (8) isconducted, a tool such as a needle-like member or an edge-like member,whereby perforation or cutting is made easy (not shown in figures), maybe installed at an inner wall of the cover (2) if necessary. On theother hand, in an embodiment where the disinfectant/bactericide (9) isreleased by breaking the second bag-shaped member (8), it is preferredin terms of ease of the breakage that the second bag-shaped member (8)be composed of a hard material such as glass and hard plastics.

In the structure where a substantially tight-sealed system can bemaintained and a sufficient breaking force from outside can be appliedto the bag-shaped member, it is possible to easily apply a strongtension to such a hard material and to break it, whereby the contentscan be removed therefrom. An example of the preferred structure is thata strong tension is concentrated on a specific area (that may be two ormore areas but a considerably narrow area is preferred) of thebag-shaped member of a hard material. As mentioned in detail byreferring to FIGS. 6 to 16 hereunder, it is to be understood that such athing can be achieved by various structures and it is also possible toadopt a structure where known structure and conventional structure areapplied to the microorganism-detecting apparatus, or installed withinthe microorganism-detecting apparatus of the present invention. Theforce from outside may be that utilizing rotational movement, pushing-inmovement, twist, etc.

Rod-Like Material

The rod like material (stem) (6 a) of the microorganism-collecting partin the present invention is a material having a so-called auxiliaryfunction, whereby a microorganism-collecting end (6 b) having a functionof direct collection of the microorganism to be tested can be stablysupported avoiding a contamination and, in addition, themicroorganism-collecting end (6 b) is kept at a position suitable forcontacting it with the medium (3) upon incubation of the microorganism.There is no particular limitation for the material, length, shape, etc.of the rod-like material (6 a) so long as such functions of a stableholding and of a security for the contacting position are substantiallyachieved. In terms of prevention of contamination and softness uponcollection of the microorganism, it is preferred that the rod-likematerial (6 a) be composed of a flexible plastic.

Microorganism-Collecting End

In the present invention, the microorganism-collecting end (6 b) ofmicroorganism-collecting part is of a material such that it directlycollects the microorganism, is capable of receiving the collectedmicroorganism in a receiving container without contamination aftercollection of the microorganism, and also contacts with the medium (3)during incubation of the microorganism so that a condition suitable forincubation of the collected microorganism is provided. So long as suchcollecting and contacting functions are substantially achieved, there isno limitation for its structure but any structure will do and there isno particular limitation for the material, length, shape, etc. of themicroorganism-collecting end (6 b). To be more specific, themicroorganism-collecting end (6 b) may be, for example, just an end ofthe rod-shaped element (6 a). However, for an object of easily achievingthe above-mentioned collecting and contacting functions, it is preferredthat the microorganism-collecting end (6 b) has a an increased surfacearea.

Preferred examples of the “increased surface area” are a surface havingone or more concavities (cutting, uneven surface, etc.) (such as an endlike an ear pick or spatula) and a porous surface. In an embodimentwhere the microorganism-collecting end (6 b) (at least a surface areathereof) is composed of a porous material, a porous material in a formof xerogel or a fibrous material is preferably used.

With regard to the fibrous material, filter paper, cottom (such asabsorbent cotton), punch felt, cloth, knitted goods, nonwoven fabric,etc. may be used without particular limitation. It is also possible touse a structure where rubber or plastic material is used partially, ahollow structure or a plunger structure using an elastic material or aflexible material or a suckable structure together with a rod-shapedelement (6 a) which constitutes a microorganism-collecting part, wherebythe sample can be easily collected when it is liquid. Particularlypreferred is one in a shape of an applicator having cotton at its end.

Examples of the object (an object to be tested) wherefrom themicroorganism is collected are usually utensils of cuisine, cookingdevices, etc., home kitchen, toilet, bathtub, the place where food andbeverage are sold, etc. Such an object is, for example, wiped with anddipped into the end of the applicator (microorganism-collecting end (6b)) as mentioned above. It goes without saying that, in that case,collection of the sample is usually conducted making sure that theapplicator does not touch an unnecessary part or area. Other examples ofthe object wherefrom the microorganism is collected are food and foodmaterials.

Medium

There is no particular limitation for the type, composition, etc. of theabove-mentioned medium (3) so long as incubation of the expectedmicroorganism to be detected (such as food poisoning microorganism orresistant microorganism) is possible, and known medium for such amicroorganism or an improved medium thereof as well as a medium which ismodified for better usability may be used. Preferably in the case of afood poisoning microorganism, it is possible to suitably select themedium by taking the compatibility (such as reactivity, intersolubility,etc.) with the later-mentioned antibiotic substances, pH indicator, etc.into consideration.

With regard to a medium for Staphylococcus aureus, it is possible to usemannitol-salt (modified) medium, Baird-Parker medium, tellurite-glycinemedium, phenylethanol-azide medium, chocolate-agar medium, blood-agarmedium, heart infusion agar medium, etc. in the present invention and,in view of the adaptability with the later-mentioned antibioticsubstance for selecting Staphylococcus aureus, the use of amannitol-salt (modified) medium is preferred.

As a medium for microorganism resistant to antibiotics such as MRSA,that which is prepared by adding oxacillin, aztreonam, polymyxin B, orthe like to the afore-described medium may be preferably used.

As a medium for Vibrio parahaemolyticus, it is possible to usesalt-polymyxin (modified) medium, cellobiose-polymyxin-colistin medium,thiosulfate-citrate-bile salts-sucrose medium, TCBS medium, MacConkeyagar medium, blood-agar medium, etc. and, in view of the adaptabilitywith the later-mentioned antibiotics for selecting Vibrioparahaemolyticus, the use of a salt-polymyxin (modified) medium ispreferred.

As a medium for Salmonella, it is possible to use xylose-lysine(modified) medium, mannitol-lysine-Crystal Violet Brilliant medium,Salmonella shigella medium, deoxycholate-citrate-lactose-sucrose medium,DHL agar medium, MacConkey agar medium, etc. and, in view of theadaptability with the later-mentioned antibiotics for selectingSalmonella, the use of a xylose-lysine (modified) medium is preferred.

As a medium for Escherichia coli, it is possible to use BTB-lactose-agarmedium, blood-agar medium, deoxycholate medium, LB medium, etc.

With regard to such a medium, it is possible to select from thosementioned in known literatures or from those prepared bymodifying/varying the above-mentioned ones. Examples of the knownliteratures are Examined Japanese Patent Publication Hei-07/73,509 (JPHei 07/73,509 B), Unexamined Japanese Patent Publication (Laid-Open)Hei-01/296,998 (JP Hei 01/296,998 A), etc. for a medium for Vibrioparahaemolyticus; Laid-Open Japanese Patent Publications Sho-52/134,082(JP Sho 52/134,082 A), Hei-06/217,760 (JP Hei 06/217,760 A), etc. for amedium for Staphylococcus aureus; Laid-Open Japanese Patent PublicationHei-02/65,798 (JP Hei 02/65,798 A), Hei-05/130,859 (JP Hei 05/130,859A), Hei-06/22,791 (JP Hei 06/22,791 A), etc. for a medium forSalmonella; etc. as well as the references cited therein. In the presentinvention, a medium which is optionally modified/changed for adaptingespecially as a liquid medium is preferred.

Usually, a medium in a liquid state is preferably used and that filledor received in a tightly-sealable containers such as ampules or capsulesis suitably used although it is not limited thereto but, for example, itmay by in such a form that it can be supplied to an incubating space ofthe container separated by a partition or the like which can be easilybroken by a force from outside.

The medium may be an agar medium and, for example, it may be in a formthat it does not contact an antibiotic substance, being separated by apartition or the like which can be easily broken by a force fromoutside. It may be in such a form that, upon incubation, the partitionis broken by pushing with an end of the microorganism-collecting rodconstituted by a hard material; it is broken by hitting an iron piece orthe like coated with glass, plastics or the like placed on the partitionby means of a magnet placed outside; or, in addition to the above, acapsule of an antibiotic substance solution or a buffer solution isbroken to flow the solution on a medium whereby the microorganism at thecollecting end, the antibiotic substance and the agar medium arecontacted. It may also be in such a form that dry medium or powdermedium is received therein instead of the agar medium and isreconstituted to the conventional agar medium using an aqueous liquidsuch as a buffer solution which is received in various forms in thecontainer of the microorganism-detecting apparatus.

In the case of the food poisoning microorganism, it is possible todetect and identify the microorganism by incubating as colonies (bymeans for a medium in a form of gel, for example). However, in aviewpoint of easiness of detection and identification of the foodpoisoning microorganism in usual restaurants and home, it is preferredto use a liquid medium giving a homogeneous incubating system to which apH indicator or certain chemical substance capable of acting as anindicator after converted to decomposed product or metabolite as aresult of decomposition or metabolism by the microorganism forproliferation of the microorganism is added whereby the judgment can beeasily conducted by means of changes in color due to pH change or asviewed by the naked eye. The medium (3) may preferably be somewhatviscous so long as the detection/identification of the food poisoningmicroorganism by means of color change is not substantially disturbed.

Additives

If necessary, various additives may be added to the above-mentionedmedium (3). In an embodiment of the present invention where detection ofthe microorganism to be detected is confirmed by means of pH change dueto proliferation of the microorganism, it is preferred that a pHindicator is previously added to the medium. When such a pH indicator isused, pH of the medium changes as a result of production of acidicsubstances (such as lactic acid) by proliferation of the microorganismwhereby the color tone of the indicator changes and, accordingly,confirmation of the proliferation becomes easy.

There is no particular limitation for the pH indicator which isapplicable to the present invention. In view of pH change region, colortone, etc., Phenol Red, Cresol Red, Bromthymol Blue, Bromcresol Purple,triphenyltetrazolium, Blue Tetrazolium, etc. can be appropriately used.Such pH indicators may be used either solely or jointly by combining twoor more if necessary. It is also preferred to use each of the pHindicators having different color tones (before changed) in each of themedia for detecting different types of microorganisms because themicroorganism to be detected can be easily recognized at a glance bymeans of such color tones.

Antibiotic Substances

In the present invention, it is also possible that the medium itself isbestowed with some selectivity to the microorganism but, usually, inorder to make the selectivity sure, it is preferred that an antibioticsubstance which effectively inhibits the proliferation of themicroorganism other than the specific one to be detected and identifiedis jointly used upon necessity. This antibiotic substance is to achievea selective incubation of the microorganism to be detected and has afunction of inhibiting the proliferation of a microorganism which isother than the specific one to be detected. The type of the antibioticsubstance used therefor may be selected depending upon the relation withthe microorganism to be detected or may be selected by considering whatkind of microorganism selectivity is to be bestowed depending upon thestate upon actual use of the apparatus of the present invention.

The “selectivity” upon incubation of the microorganism can be adjustedor selected depending upon the purpose. For example, when a foodpoisoning microorganism is an object, it is preferred to have anincubation selectivity for one or more microorganisms selected from thegroup consisting of “Staphylococcus aureus, Vibrio parahaemolyticus andSalmonella” which occupy nearly all (around 99%) of the microorganismscausing food poisoning, while discrimination among those threemicroorganisms is not essential. However, from a viewpoint of a minutecountermeasure to each of the characteristics of those food poisoningmicroorganisms, it is in some cases preferred to have an “incubationselectivity” whereby discrimination among “Staphylococcus aureus, Vibrioparahaemolyticus and Salmonella” is possible. In addition, there is acase where it is preferred to have an “incubation selectivity” wherebyenteropathogenic Escherichia coli such as E. coli O-157 can bediscriminatively detected.

In the present invention, there is no particular limitation for theantibiotic substance to be used so long as it is capable of effectivelyinhibiting a microorganism other than specific (one or more)microorganism(s) to be detected such as a food poisoning microorganism.Although the antibiotic substance may be used solely in some cases,preferably, two or more may be used jointly depending upon the purpose.The amount may be suitably decided especially depending upon the amountof the medium used. For example, in detecting a food poisoningmicroorganism, the following antibiotic substances are preferably used.In the following description, the concentration for each antibioticsubstance is a suitable concentration at the incubating stage of thefood poisoning microorganism (when unitedly used with the medium).

<Antibiotic Substances Selective to Staphylococcus aureus> Aztreonam 1to 15 μg/ml Polymyxin B 1 to 15 μg/ml Fluconazole 1 to 10 μg/ml

Combination of the above three antibiotic substances can be usedpreferably.

<Antibiotic Substances Selective to Vibrio parahaemolyticus> Polymyxin B1 to 15 μg/ml Fluconazole 1 to 10 μg/ml Potassium tellurite 1 to 20μg/ml

Combination of the above three antibiotic substances can be usedpreferably.

<Antibiotic Substance Selective to Salmonella> Fluconazole 1 to 10 μg/ml

Locating Position of the Antibiotic Substances

There is no particular limitation for the position or the place wherethe antibiotic substances are located or placed so long as they functionunitedly with the medium (3) at the incubating stage of themicroorganism. Preferably, it can be held at a disk member (7) as willbe mentioned later in detail (cf. FIG. 5) while other methods arepossible as well. Thus, to be more specific, the antibiotic substancemay be previously added to a medium (3), coated on an inner wall of thecontainer or bestowed at a certain place of the microorganism-collectingpart (6) (the place being able to contact with the medium (3) uponincubation). Two or more of such positions may be applied if necessary.It goes without saying that, in those cases, the above-mentioned diskmember (7) may be omitted. When the antibiotic substance is previouslyadded to a medium (3), it is preferred, by taking inactivation of theantibiotic substance in the medium into consideration, to add theantibiotic substance in such a little amount that the inactivation(usually, a decrease in the antibiotic activity to an extent of around30 to 35% when stored at ambient temperature for one year) can besupplemented.

When an antibiotic substance is received in a space which is used forincubation in a container, the antibiotic substance may be received in apowdery form but may be received as capsules or tablets which are easilydissolved upon contacting with a liquid such as a medium or, after itssolution is added to a position to be used as a space for incubation inthe container as mentioned above, it may be received by coating on aninner wall of the container by means of a vacuum drying or the like.Alternatively, the antibiotic substance in a form of an easily solublepowdery or a solution may be filled in a breakable ampule or capsule andreceived in a space used for incubation in the container in such amanner that it can be easily added. It may be received in any place ofthe container and may be received in such a manner that it can contactwith the medium by shaking the container, by washing out with a liquidmedium or by breaking the ampule by a force from outside beforeincubation. A preferred receiving method is that, before incubation, itkeeps a noncontact state with the medium and, for example, theantibiotic substance may be placed at the partition member (5) or abovethe guide member (20).

On the other hand, in an embodiment where the antibiotic substance islocated at a certain position of the microorganism-collecting part (6),it is preferred that the antibiotic substance is located at therod-shaped member (6 a) (the position where contact with the medium (3)is possible) of the microorganism-collecting part (6) and/or at themicroorganism-collecting end (6 b) which is an end of themicroorganism-collecting part (6). When the antibiotic substance isplaced at the microorganism-collecting end (6 b), it may be impregnatedinto the microorganism-collecting end (6 b) itself or, after preparing amulti-layered structure consisting of an inner layer (at the side of therod-shaped element (6 a) which is a microorganism-collecting end (6 b)and an outer layer (for collecting the microorganism), the antibioticsubstance is placed on the inner layer. Alternatively, the antibioticsubstance may be impregnated in or coated on the upper half area of themicroorganism-collecting end (6 b).

Disk Member

A disk member (7) which is optionally used in the present invention isof a material having the functions of holding the antibiotic substanceand for offering the condition suitable for incubation of the specifickind of microorganism upon contacting with the medium (3) during theincubation (cf. FIG. 5). There is no particular limitation for thematerial, length, shape, etc. of the disk member so long as such holdingand contacting functions are substantially achieved. To be morespecific, the disk member may be a simple board or a flat material but,in view of an easy and effective achievement of the above-mentionedholding and contacting functions, it is preferred that the disk memberhas an increased surface area. Examples of such “an increased surfacearea” are a surface with one or more concavities (cutting, unevensurface, etc.) and a porous surface.

In an embodiment where the disk member (at least its surface area) iscomposed of a porous material, the preferably used one is a xerogel-likeporous material or a fibrous material. Examples of the fibrous materialwhich are applicable without limitation are filter paper, cotton (suchas absorbent cotton), punch felt, cloth, knitted goods, nonwoven fabric,etc.

Examples of a medium which does not need a disk member (7) for holdingthe antibiotic substance, improves the operation ability of themicroorganism-detecting apparatus, and is suitable as a liquid medium aswell, are as follows:

(a) Composition of a Medium for Salmonella (per 1,000 ml of the medium):

An appropriate amount of tryptone, preferably 3 to 7 g, for instance, 5g; an appropriate amount of yeast extract, preferably 1 to 6 g, forinstance, 3 g; an appropriate amount of lysine, preferably 5 to 15 g,for instance, 10 g; an appropriate amount of glucose, preferably 0.5 to2 g, for instance, 1 g; an appropriate amount of sodium chloride,preferably 7 to 9 g, for instance, 8 g; an appropriate amount ofmonopotassium dihydrogen phosphate, preferably 1.0 to 2.0 g, forinstance, 1.6 g; an appropriate amount of sodium thiosulfate, preferably0.1 to 0.3 g, for instance, 0.2 g; an appropriate amount of ammoniumiron citrate, preferably 0.2 to 0.4 g, for instance, 0.3 g; anappropriate amount of magnesium chloride, preferably 15 to 25 g, forinstance, 20.3 g; an appropriate amount of 0.4% Malachite Greensolution, preferably 27 to 33 ml, for instance, 30 ml; and anappropriate amount of Bromcresol Purple, preferably 0.01 to 0.03 g, forinstance, 0.02 g

per 1,000 ml of the medium (pH of the medium being about 5.3 to 5.7);

(b) Composition of a Medium for Vibrio such as Vibrio parahaemolyticus(per 1,000 ml of the medium):

An appropriate amount of a salt polymyxin broth, for instance, 25 to 40g (where a salt polymyxin broth contains an appropriate amount of yeastextract, an appropriate amount of peptone, an appropriate amount ofsodium chloride and an appropriate amount polymyxin B); an appropriateamount of mannitol, preferably 15 to 25 g, for instance, 20 g; anappropriate amount of sodium citrate, preferably 5 to 10 g, forinstance, 8 g; an appropriate amount of sodium thiosulfate, preferably0.1 to 0.3 g, for instance, 0.2 g; and an appropriate amount ofBromocresol Purple, preferably 0.01 to 0.03 g, for instance, 0.02 g

per 1,000 ml of the medium (pH of the medium being about 7.0 to 7.4);

(c) Composition of a Medium for Escherichia coli (per 1,000 ml of themedium):

An appropriate amount of lauryl sulfate broth, preferably 26 to 43 g,for instance, 35.6 g (the lauryl sulfate broth contains an appropriateamount of tryptose, an appropriate amount of lactose, an appropriateamount of monopotassium dihydrogen phosphate, an appropriate amount ofdipotassium monohydrogen phosphate, an appropriate amount of sodiumchloride and an appropriate amount of sodium lauryl sulfate); anappropriate amount of lactose, preferably 3 to 8 g, for instance, 5 g;and an appropriate amount of Bromthymol Blue or Bromocresol Purple,preferably 0.03 to 0.05 g, for instance, 0.04 g

per 1,000 ml of the medium (pH of the medium being about 6.75 to 7.25);and

(d) Composition of a Medium for Staphylococcus such as Staphylococcusaureus (per 1,000 ml of the medium):

An appropriate amount of tryptone, preferably 5 to 15 g, for instance,10 g; an appropriate amount of yeast extract, preferably 2 to 8 g, forinstance, 5 g; an appropriate amount of mannitol, preferably 5 to 15 g,for instance, 10 g; an appropriate amount of dipotassium monohydrogenphosphate, preferably 2 to 8 g, for instance, 5 g; an appropriate amountof lithium chloride, preferably 5 to 6 g, for instance, 5.5 g; anappropriate amount of glycine, preferably 12 to 20 g, for instance, 16.5g; an appropriate amount of sodium pyruvate, preferably 10 to 14 g, forinstance, 12 g; an appropriate amount of 1% aqueous potassium telluritesolution, preferably 12 to 18 ml, for instance, 15 ml; and anappropriate amount of Phenol Red, preferably 0.02 to 0.03 g, forinstance, 0.025 g

per 1,000 ml of the medium (pH of the medium being about 7.25 to 7.75).

Among the components for the medium, amounts of those which are added asnutrients such as tryptone, yeast extract, lysine, glucose, peptone,mannitol, tryptose and lactose may be either increased or decreased bypersons skilled in the art depending upon the object and necessity, andit is to be understood that such a medium is also included in thecoverage of the medium of the present invention. Examples of thecomponents related to the selectivity of the microorganism to bedetected are sodium chloride, Malachite Green, Bromcresol Purple, sodiumthiosulfate, ammonium iron citrate, polymyxin B, sodium lauryl sulfate,Bromthymol Blue, lithium chloride, sodium pyruvate, potassium telluriteand Phenol Red. Since such components have considerable affects on thesensitivity of the microorganism detection, it is requested that theadding amounts are precisely selected. However, it is also acceptable toselect the amounts which are out of the above-mentioned ranges afterconducting normal tests or the like by incubating the microorganismknown in the art and selecting and deciding the due amounts. It is to beunderstood that the medium with such a modification or improvement isalso within a coverage of the present invention.

Those media are liquid ones having a fluidity and are particularlysuitable for utilization in the microorganism-detecting apparatus of thepresent invention. Those media show excellent microorganism-detectingsensitivity and specificity to the microorganism and, at the same time,they have an excellent detectability of the microorganism due to colorchange of the pH indicator. The amounts of the components constitutingthe above-mentioned media may be either increased or decreased by aperson skilled in the art and it is also possible to add appropriatecomponent(s) provided that the basic characteristics are unaltered.Commercially-available ones may be used as the components to becompounded with the medium. For example, salt-polymyxin broth isavailable from NISSUI SEIYAKU K.K., Japan, lauryl sulfate broth isavailable from DIFCO and other components are available from DIFCO, WAKOPURE CHEMICAL INDUSTRIES, LTD., etc.

The apparatus of the present invention for the detection ofmicroorganisms is useful for detecting specific kinds of microorganisms(food poisoning microorganisms such as Staphylococcus aureus,Salmonella, Escherichia coli, Vibrio parahaemolyticus and otherintestinal microorganisms) in cooking environments (such as equipmentand utensil at cuisine) and human living environments including kitchen,toilet, bath room and shops where food and beverage are sold. It is alsopossible to utilize the microorganism-detecting apparatus of the presentinvention with an object of detecting the afore-described microorganismsin food and food materials containing various components in highconcentrations such as for detecting the contamination by food poisoningmicroorganisms. Food and food materials contain various components suchas saccharides, proteins, lipids, organic acids (e.g., acetic acid andcitric acid), amino acids, chemical condiments, vitamins, minerals andother chemical substances. As a result thereof, pH, ion strength, etc.of the medium change and, when detection is conducted where theprinciple of the detection utilizes the color change of the pH indicatorfor the pH change of the medium due to proliferation of microorganisms,there is a possibility that the above changes in pH and ion strengthaffect on the pH indicator used there. In that case, it is possible toprepare an appropriate sample depending upon the type of the food andfood materials to be tested by, for example, subjecting the sample to apretreatment (such as adjustment of pH), selecting an appropriate pHindicator, changing the components of the medium or changing the amountsof the components. For example, when the food to be tested is a beveragesuch as a milk product and an object is to detect Escherichia coli,Bromthymol Blue used as a pH indicator may be substituted withBromcresol Purple.

Aseptic Condition

The microorganism-detecting apparatus of the present invention asmentioned above is preferably in an aseptic condition (at least in itsinner area) immediately before the use in view of correctness of thetest. In making the apparatus of the present invention aseptic after itsmanufacture, known physical measures (e.g., sterilization by irradiationof electromagnetic waves such as ultraviolet ray and gamma-ray) and/orchemical measures (e.g., sterilization by gas such as ethylene oxide)may be used without particular limitation. For example, the apparatuscan be sterilized by irradiating with gamma-ray at 1 to 30° C. for oneminute. When sterilization is conducted by a sterilizer using ethyleneoxide, sterilization can be achieved by conducting at, for example, 0 to70° C. (preferably, at 30 to 50° C.) in a gas consisting of about 10 to40%; of ethylene oxide and about 60 to 90% of carbon dioxide gas(preferably, about 20% of ethylene oxide and about 80% of carbon dioxidegas) under certain pressure (preferably, at the pressure of about 0.1 to1 kgw/m² or, more preferably, at about 0.3 kgw/m²) for about 1 to 48hours (preferably, about 3 to 10 hours).

After the above-mentioned sterilization, it is preferred that themicroorganism-detecting apparatus of the present invention is stored ina bag which is made of film or the like (not shown). At that time, themicroorganism-detecting apparatus (FIG. 1) may be unitedly stored in thesame bag or, if necessary, each of the container and the cover (2) (andthe microorganism-collecting part (6)) may be stored in differentenclosures.

With regard to selection and combination of the afore-describedmicroorganism-collecting end, various receiving forms concerning theantibiotic substances and various forms concerning the medium, a lot ofvariations and combinations are allowable provided that they are not outof the objects and characteristic features of the present invention.Especially in the case where both (or at least one of) antibioticsubstance and medium (particularly preferably, liquid medium) are/isreceived in a bag, ampule or capsule made of synthetic resin or glassand, upon actual use, the receiver is broken whereby the antibioticsubstance and the medium are unitedly combined, it is preferred that thearea of the container receiving the bag, ampule or capsule is made of amaterial or in a shape whereby such bag, ampule or capsule can be easilybroken or destroyed by a breaking device or by a force from outsidewithout such a device. For example, the area is flexible or is asubstance having elasticity or flexibility.

Disinfectant/Bactericide

The disinfectant or bactericide which is used for disinfecting and/orsterilizing the incubated pathogenic microorganism in themicroorganism-detecting apparatus after use, whereby the usedmicroorganism-detecting apparatus can be safely disposed is added insuch a state that it is substantially separated from the outside or fromthe environment once the incubation of the microorganism to be tested isstarted so that the area containing the incubated microorganism can bedisinfected and/or sterilized. If it is in such a structure, it may belocated in the microorganism--detecting apparatus of the presentinvention in any form but a representative example is thatdisinfectant/bactericide is present in a form that it is received in thesecond bag-shaped member (8) which is different from the firstbag-shaped member for receiving the medium (3) in a cover (2). Withregard to the disinfectant/bactericide, one which is suitably selectedfrom disinfectants and/or bactericides which have been known in the artmay be used and the examples are disinfectants/bactericides of achlorate type, those of an invert soap type, those of a biguanide type,those of an aldehyde type and those of a phenol or cresol type.Representative examples of the disinfectant/ bactericide are sodiumhypochlorite, bleaching powder, chloramine-T, benzalkonium chloride,benzethonium chloride, chlorhexidine salt, polyalkylene biguanidinesalt, formaline, glutaraldehyde and pompidon iodine.

The disinfectant (bactericide) used may be selected from those whichhave been commonly used for disinfecting, sterilizing or washing thearea wherefrom pathogenic microorganisms such as food poisoningmicroorganisms were detected, such as a chopping board, and cookingboard or which have been found to be preferred for such an object.Examples of such a detergent (disinfectant) are the above-mentionedsodium hypochlorite (WAKO PURE CHEMICAL INDUSTRIES, LTD., Japan),“Tego-51” (10% ; trade name of NIPPON SHOJI K.K., Japan), “KitchenKabi-Killer” (trade name of JOHNSON K.K., Japan) and “Kitchen Haiter”(trade name of KAO K.K., Japan).

Such a detergent (disinfectant) may also be that where its disinfectingeffect has been well noted by the following test method using, forexample, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922,Enterobacter faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853.

Method for Testing the Disinfecting (Sterilizing) Effect

(1) Preparation of Microorganism Solution

Standard strains of Staphylococcus aureus ATCC 25923, Escherichia coliATCC 25922, Enterobacter faecalis ATCC 29212 and Pseudomonas aeruginosaATCC 27853 incubated for 24 hours were used and prepared into a solutionhaving a McFarland's standard turbidity of No.1 (3.0×10⁸ CFU/ml) using asterile physiological saline solution.

(2) Sterilizing Method

Each of the washings was used as an original solution and contacted, ina final concentration of 10%, for five minutes with the microorganismsolution prepared in the above (1).

(3) Incubating (Culturing) Method

The microorganism solution contacted with the washing for five minutesas mentioned in the above (2) was used as an original solution, dilutedto 10-fold (×10) and 100-fold (×100) with a sterile physiological salinesolution and each 0.1 ml of it was inoculated to a blood-agar mediumfollowed by incubating. As a control, a system containing no detergentwas prepared and incubated by the same manner as above. Numbers of thecolonies grown after incubating at 37° C. for 48 hours were comparedwith those of the control whereby the disinfecting effect was evaluated.

When sodium hypochlorite (WAKO PURE CHEMICAL INDUSTRIES, LTD., Japan),“Tego-51” (10% ; trade name of NIPPON SHOJI K.K., Japan), “KitchinKabi-Killer” (trade name of JOHNSON K.K., Japan) and “Kitchen Haiter”(trade name of KAO K.K., Japan) were used as detergents, all of thestandard strains were found to be sterilized within five minutes bythose detergents.

FIG. 4 shows another embodiment of the microorganism-detecting apparatusof the present invention. In this embodiment of FIG. 4, a “guide member”(20) has a function of promoting the falling-down of a medium (3) alongthe rod-shaped element (6 a) when the medium (3) falls down into acontainer (1), and the guide member is disposed at the lower area of thepartition member (5). Constitution of FIG. 4 is the same as that of FIG.1 except that the guide member (20) is installed therein.

There is no particular limitation for the material shape, size, etc. ofthe guide member (20) so long as the guide member (20) plays a functionof promoting the falling-down of the medium (3) along the rod-shapedelement (6 a). In view of the easiness in promoting the falling-down ofthe medium (3) along the rod-shaped element (6 a), it is preferred thatthe guide member (20), at least a part thereof, has a smaller innerdiameter than that of the container (1).

To be more specific, it is preferred that, as shown in FIG. 4, the guidemember (20) is composed of a “fan”-shaped member where the lower end ofthe guide member is somewhat inclined to the central direction of thecontainer (1) (i.e., in other words, the lower end has a smallerdiameter than the inner diameter of the container (1)) although such an“inclination” may be omitted. FIG. 5 shows a microorganism-detectingapparatus of the present invention where the disk member (7) is placedin the container (1).

FIGS. 6 to 11 show a basic constitution of the microorganism-detectingapparatus of the present invention in another representative embodiment,particularly to show a constitution basically bestowed with structurefor disinfecting and/or sterilizing the cultured medium, etc. aferincubation. FIG. 6 mainly sows a cross sectional view of themicroorganism-detecting apparatus; FIG. 7 mainly shows an outerappearance of the microorganism-detecting apparatus; FIG. 8 shows anouter appearance of the container (21) for the microorganism-detectingapparatus; FIG. 9a shows a view from the upper side of the container(21) for the microorganism-detecting apparatus; FIG. 9b shows a crosssectional view of the side of the container (21) for themicroorganism-detecting apparatus; FIG. 10a is a shape of the cover (22)for the microorganism-detecting apparatus and is the shape observed fromthe connected side with the container (21); FIG. 10b mainly shows anouter appearance from one side of the cover (22) for themicroorganism-detecting apparatus; FIG. 10c is a shape of the cover (22)for the microorganism-detecting apparatus and shows the view from theupper part (from the side of the cap (261)); and FIG. 11 shows the crosssection view from the side of the container for themicroorganism-detecting apparatus (the microorganism collecting part(member) is omitted in FIG. 10 and FIG. 11).

In FIG. 6, the cover (22) is mainly composed of (i) a cover-constitutingmember (housing) (23) capable of receiving an ampule (24) in which amedium is contained, (ii) a cover-constituting member (housing) (27)capable of receiving an ampule (28) in which a disinfectant iscontained, and (iii) a cap member (261). A protrusion (or projection)(32) is disposed at the bottom side of the cover-constituting member(23), i.e. at the connecting side with cylindrical container (21). Theprotrusion (32) may be placed throughout the circumference of thecover-constituting member (23) whereby the connecting side portion ofthe cover-constituting member (23) with the container (21) becomesthick. Alternatively, the protrusion (32) may be sporadically placed ina projecting manner on the inner side of the cover-constituting member(23) such as a projection of a crosspiece (sash bar) or the like. In apreferred embodiment, a screw cutting is done at the top of thecover-constituting member (23), i.e. at the end (34) at the side of thecap-material (261) as to engage with the screw threads made on thecover-constituting member (27) and, when the main body of thecover-constituting member (27) is rotated, a bottom (35) of the areawhere the ampule (28) in the cover-constituting member (27) is receivedcan be pushed against the side of the container (21). Alternatively, thetop of the cover-constituting member (23), i.e. an end (34) at the sideof the cap member (261), is tightly engaged with an end of thecover-constituting member (27) at the side of the container (21) and,when the cover-constituting member (27) is pushed by sliding to the sideof the container (21), the bottom (35) of the ampule (28)-receiving areaof the cover-constituting member (27) can be easily pushed against theside of the container (21) whereby the ampule (24) can be pushed againstthe side of the container (21).

In the cover-constituting member (27), there are through holes at thebottom (35) of the receiving part for ampules (28) so that thedisinfectant in the ampule flows down into the container (21) when theampule (28) is broken. Like in the cover-constituting member (23), thereare protrusions (or projections) (33) at the bottom side of thecover-constituting member (27), i.e. at the side of the ampule (24).Like in the afore-mentioned protrusions (32), the protrusions (33) maybe placed throughout the circumference of the cover-constituting member(27) whereby the bottom (35) of the ampule-receiving part of thecover-constituting member (27) becomes thick at the side of the ampule(24). Alternatively, the protrusions (33) may be sporadically placed ina projecting manner on the inner side of the cover-constituting member(27). In a preferred embodiment, a screw cutting is done at the top ofthe cover-constituting member (27), i.e. at the end (37) at the side ofthe cap-material (261) so as to engage with the screw threads made onthe cover-constituting member (261) and, when the head of the cap member(261) is rotated, a bottom (36) of the cap member (261) can be pushedagainst the side of the ampule (28). Alternatively, the top of thecover-constituting member (27), i.e. an end (37) at the side of the capmember (261), is tightly engaged with the cap member (261) and, when thecap member (261) is pushed into by sliding to the side of the container(21), the bottom (36) of the ampule (28)-receiving area of thecover-constituting member (261) can be easily pushed against the side ofthe container (21) whereby the ampule (28) can be pushed against theside of the container (21).

First, when the main part of the cover-constituting member (27) isrotated (or pushed against the container side) and the bottom (35) ofthe receiving area for the ampule (28) in the cover-constituting member(27) is pushed against the side of the container (21), the bottom (35)pushes down the ampule (24). Such a pushed-down ampule (24) is thensupported with the slope of the protrusion (32) disposed on thecover-constituting member (23). As such, when the cover-constitutingmember (27) is pushed down to the side of the container (21), a tensionwould occur at a contacting point (A) of the ampule (24) with the slopeof the protrusion (32) whereby the lower part of the ampule (24) isbroken and the contents therein is allowed to be released. The contentscoming out from the ampule (24) flows down into a container (21) due togravity and contacts with the microorganism-collecting end (26 b).(Incidentally, in FIG. 6, through holes, attaching area of themicroorganism-collecting part, fine structure of joint portions betweenthe cover and the container, etc. are omitted.)

During the disinfection after incubation of the microorganism, when thetop of the cap member (261) is rotated (or pushed against the side ofthe container) and the bottom (36) of the cap member (261) is pushedagainst the side of the ampule (28), the bottom (36) of the cap member(261) pushes down the ampule (28). The pushed-down ampule (28) is thensupported on the slope of the protrusion (33) placed at thecover-constituting member (27). As such, when the cap member (261) ispushed against the side of the container (21), a tension would occur ata contacting point (B) of the ampule (28) with the slope of theprotrusion (33) whereby the lower part of the ampule (28) is broken andthe contents therein is allowed to be released. The contents coming outfrom the ampule (28) flows down into a container (21) through theperforations placed at the bottom (35) of the ampule (28)-receiving partof the cover-constituting member (27) due to gravity and contacts theincubated microorganism. It is preferred in view of the actual use thatthe ampule is made of a fragile material such as glass or hard plasticand that, when a mechanical force is applied thereto (particularly whenthe mechanical force is partly applied), the ampule is easily broken.

The medium containing the incubated pathogenic microorganism and thespace in the container where there is a high possibility ofcontamination with the microorganism are disinfected and/or sterilizedby a disinfectant. As such, the apparatus can be made into a safelydisposable state. It is possible for the incubation system of theapparatus of the present invention that, since the beginning ofincubation by supplying the medium into an apparatus (1), certainmicroorganisms (targets) are found/detected by observing the incubatedmedium and at last the apparatus is disinfected and/or sterilized tomake it disposable while the incubation system is kept in asubstantially tightly closed state relative to the outside or to theenvironment throughout the above steps. The microorganism-detectingapparatus of the present invention is useful with a view that even ifthe target microorganism to be tested is not limited to pathogenic butordinary (nonpathogenic), pollution of environment (or possibilitythereof) is to be avoided. Further, the microorganism-detectingapparatus has excellent portability and, therefore, the fact that theapparatus can be disinfected/sterilized to such an extent that it can besafely and surely disposed is valuable and appreciated. In addition, itis clear from the structure of the apparatus of the present inventionthat the microorganism-detecting apparatus can be surely, easily andsecurely operated even by unskillful persons.

FIG. 12 and FIG. 13 show a constitution which is fundamentally presentin another representative embodiment of the microorganism-detectingapparatus of the present invention, particularly with structure fordisinfecting/sterilizing the cultured medium, etc. after incubation.

In FIG. 13, a cover (42) is mainly composed of (i) a cover-constitutingmember (43) capable of receiving an ampule (44) containing a medium,(ii) a cover-constituting member (47) capable of receiving an ampule(48) containing a disinfectant, and (iii) a cap member (49). At thebottom side of the cover-constituting member (43), i.e. at the side ofthe junction (59) with the cylindrical container (41), there is aprotruding portion (or projection) (52). The protrusion (52) may beplaced throughout the circumference of the cover-constituting member(43) whereby the cover-constituting member (43) becomes thick at theengaging side (59) thereof with the container (41), or the protrusion(52) may be sporadically placed in a projecting manner on the inner sideof the cover-constituting member (43), such as a protrusion of acrosspiece or the like. In a preferred embodiment, a screw cutting isdone at the top of the cover-constituting member (43), i.e. at the end(54) at the side of the cap member (49) so as to engage with the screwthreads made on the cover-constituting member (47) and, when a head part(45) of the cover-constituting member (47) is rotated, a bottom (55) ofthe cover-constituting member (47) can be pushed against the side of thecontainer (41). The bottom part (55) of the cover-constituting member(47) has one or more through holes whereby, when an ampule (48) isbroken, the disinfectant contained therein can flow into a container(41) as mentioned below (incidentally, in FIG. 13, the through hole isnot shown).

A protruding portion (or projection) (53) is disposed at the bottom sideof the cover-constituting member (47), i.e. at the side of an ampule(44), like the cover-constituting member (43). The protrusion (53) maybe disposed, like the above protrusion (52), throughout thecircumference of the cover-constituting member (47) whereby thecover-constituting member (47) becomes thick at the bottom side (55) ofthe ampule side (44). Alternatively, the protrusion (53) may besporadically placed in a projecting manner on the inner side of thecover-constituting member (47). In a preferred embodiment, a screwcutting is done at the top of the cover-constituting member (47), i.e.at the end (57) at the side of the cap member (49) so as to engage withthe screw threads made on the cap member (49) and, when a head part ofthe cap member (49) is rotated, a bottom (56) of the cap member (49) canbe pushed against the side of the ampule (48).

First, when a head part (45) of the cover-constituting member (47) isrotated so that the cover-constituting member (47) is pushed against theside of the container (41), a bottom (55) of the cover-constitutingmember (47) pushes down the ampule (44). The pushed-down ampule (44) isthen supported by a slope of the protrusion (or projection) (52)disposed in the cover-constituting member (43). As thecover-constituting member (47) is pushed further against the side of thecontainer (41), a tension becomes higher at the contacting portion (A)between the ampule (44) and a slope of the protrusion (52) whereupon thelower area of the ampule (44) is broken and the content therein isallowed to release. The contents coming out from the ampule (44) flowsdown into the container (41) because of the force of gravity and comesin contact with the microorganism-collecting end (46 b). (Incidentally,in FIG. 13, a through hole at (59), a connecting area for themicroorganism-collecting member, the detailed structure of theengagement between the cover and the container, etc. are omitted.)

At the disinfecting stage after incubation, when the bottom (56) of thecap member (49) is pushed against the side of the ampule (48) byrotating the top portion (461) of the cap member (49), a bottom (56) ofthe cap member (49) pushes down the ampule (48). The pushed-down ampule(48) is then supported by a slope of the protrusion (53) disposed in thecover-constituting member (47). As the cap member (49) is pushed furtheragainst the container (41) side, a tension would occur higher at thecontacting area (B) of the ampule (48) with the slope of the protrusion(53) whereupon the lower part of the ampule (48) is broken and thecontents therein is allowed to release. The contents coming out from theampule (48) flows down by gravity into a container (41) through aperforated hole (through hole) (not shown) formed in the bottom (55) ofthe cover-constituting member (47) and comes in contact with theincubated microorganism. It is preferred in terms of actual use that theampule is made of fragile material including glass, hard plasticmaterial, etc. whereby it can be easily broken upon application(especially, partial application) of mechanical force.

The medium containing the incubated pathogenic microorganisms, etc. andthe space of the container having a high possibility of contaminationwith the microorganisms are disinfected and/or sterilized by adisinfectant. As such, the apparatus can be made safely disposable. Itis possible for the incubation system of the apparatus of the presentinvention that, since the beginning of incubation by supplying themedium into an apparatus (1), a certain microorganism is found/detectedby observing the incubated (cultured) medium and, at last, the apparatusis disinfected/sterilized to make it disposable while the incubationsystem is kept in a substantially tightly closed state relative to theoutside (surrounding atmosphere) or to the environment throughout theabove steps. The microorganism-detecting apparatus of the presentinvention is useful and characteristic with a view that themicroorganisms to be tested are not limited to pathogenic ones, andpollution of the environment (or possibility thereof) is to be avoidedeven if the microorganisms to be tested are ordinary ones. Further, themicroorganism-detecting apparatus has an excellent portability and anunique arrangement and, therefore, the fact that the apparatus can bedisinfected/sterilized to such an extent that it can be safely andsurely disposed is valuable and highly appreciated. In addition, it isclear and apparent from the arrangement of the apparatus according tothe present invention that the microorganism-detecting apparatus can besurely, securely and readily operated even by unskillful persons.

FIG. 14 shows a constitution which is fundamentally present in onerepresentative embodiment of the microorganism-detecting apparatus ofthe present invention, like those as shown in FIG. 12 and FIG. 13. Ituniquely has structure for disinfecting and/or sterilizing the culturedmedium, etc. after incubation.

In FIG. 14, the cover is mainly composed of a cover-constituting member(62) which is capable of receiving and holding (i) an ampule containinga medium and (ii) an ampule containing a disinfectant. At the sidecontacting with the surface of ampules (64/68), protrusions (orprojections) (73 and 74) are installed in a form of grooves (or ofridges) on the inner side of the cover-constituting member (62). Refer,for example, to FIG. 14(c). When a ring (77) (cf. FIG. 14(a)) having aslightly smaller diameter than a diameter (or outer diameter) of thecover-constituting member (62) accommodating the ampule is prepared andpassed from a top (71) of the cover-constituting member (62) to the sideof a container (61), the ring (77) smoothly passes at the area of (72)in the cover since there is no projection there but, after the area(72), tension due to the protrusions (73 and 74) is applied to theampule and forces the ampule to be broken from its side. When theprotrusions (73 and 74) are made to come closer to the ampule uponcoming nearer the container (61), then stronger tension bears on theampule as the ring (77) passes from the top (71) of the cover (62) tothe side of the container (61). Incidentally, in FIG. 14, a perforatedhole (through hole) for the medium and the disinfectant each coming outfrom each ampule (64/68) into a container (61), an area where themicroorganism-collecting part is attached, the detailed arrangement ofengaging portion between the cover and the container, etc. are omitted.

In another embodiment, the cover is mainly composed of acover-constituting member (62) capable of receiving and holding (i) anampule (64) containing the medium and (ii) an ampule (68) containing thedisinfectant as same as that mentioned hereinabove. Alternatively, it isalso possible that a ring (77) is previously installed on the area (76)of the cover (62) at the container (61) side and the ring (77) is raisedto the top (71) side of the cover (62) whereby breaking is started fromthe lower ampule (64) the same as above.

In still another embodiment, the cover is mainly composed of acover-constituting member (62) capable of receiving and holding (i) anampule (64) containing a disinfectant and (ii) an ampule (68) containingthe medium. A ring (77) prepared already is passed to a container (61)from the top (71) of the cover (62) so that the ampule (68) is brokenfrom its side. The ring (77) which was moved for breaking the ampule(68) as such is then moved to the middle portion of the cover (62) andeither stopped there or moved upward followed by taking away from thetop (71). For breaking another ampule (64), a ring (77) (hereinafter,referred to as (77′)) (not shown) is previously installed on the area(76) of the cover (62) at the side of the container (61) and said ring(77′) is raised to the top side (71) of the cover (62) whereby theampule (64) is broken from the lower part. Alternatively, it is alsopossible that the cover is mainly composed of a cover-constitutingmember (62) capable of receiving and holding (i) an ampule (64)containing the medium and (ii) an ampule (68) containing thedisinfectant and that the ring (77) and the ring (77′) are moved in areversed order as compared with the above, whereby the medium is firstsupplied to the container (61) and, after incubation, the disinfectantis supplied to the container (61).

FIG. 15 illustrates a fundamental arrangement required in arepresentative specific embodiment of the microorganism-detectingapparatus according to the present invention, particularly thefundamental arrangement for disinfecting and/or sterilizing the culturedmedium, etc. after incubation.

In FIG. 15, the cover (82) is mainly composed of a cover-constitutingmember capable of receiving and storing (i) an ampule (84) containing amedium and (ii) an ampule (88) containing a disinfectant. In thecover-constituting member (82), there are pushing-down members (91 and93) at the top (99) which is an opposite side to the container (81). Thepushing-down members (91) is equipped with a part (92) having a shapefor allowing a force to be applied to the top of the ampule (84) in astable manner. Similarly, another pushing-down member (93) is equippedwith a part (94) (in the drawing, this symbol is omitted) having a shapefor allowing a force to be applied to the top of the ampule (88) in astable manner.

First, when the pushing-down member (91) is pushed in, the ampule (84)is pushed down via the part (92). Then tension would occur at acontacting point (95) of the ampule (84) with a slope (orcrosspiece-like projection) (not shown) formed at the side near thecontainer (81) and on the inner side (ampule-receiving side) of thecover (82). The tension forces the lower part of the ampule to be brokenwhereby the medium is taken out from the ampule and comes into thecontainer (81) by gravity through one or more perforated holes (throughhole) (not shown) in the engaging portion (89). When anotherpushing-down member (93) is pushed in after incubation of themicroorganism, the ampule (88) is pushed down via the part (94)whereupon a tension would occur at a contacting point (96) of the ampule(88) with a slope (or crosspiece-like projection) (not shown) located atthe side near the container (81) and on the inner side (ampule-receivingand storing side) of the cover (82) and forces the lower part of theampule to be broken whereby the disinfectant is released from the ampuleand comes into the container (81) through the through hole (perforatedhole) (not shown) of the engaging portion (89) by gravity.

The pushing-down members (91/93) may be installed at the top (99) of thecover (82) in such a manner that they can be pushed in by a force fromoutside which is more than a certain degree. The pushing-down members(91/93) may also be engaged with the top member (99) by screw threadsand, in that case, the members (91/93) are in such a constitution thatthey can be pushed against the side of the container (81) by rotatingthem.

In any event, the constitution is to be in such a manner that, untilthere is a need of taking out the medium and the disinfectant in use ofthe microorganism-detecting apparatus according to the presentinvention, the bag-shaped members such as ampules are sustained freelyfrom being broken and releasing their contents but, once necessary, eachof the contents can be freely or optionally released therefrom.

FIG. 16 illustrates a fundamental arrangement required in arepresentative specific embodiment of the microorganism-detectingapparatus according to the present invention, particularly thefundamental arrangement for disinfecting and/or sterilizing the culturedmedium, etc. after incubation.

In FIG. 16, a cylindrical cover (102) is mainly composed of (i) acover-constituting member (112) which constitutes an almost lower halfof the cover (102) and (ii) another cover-constituting member (111)which constitutes an almost upper half of the cover (102). At least oneof the cover-constituting members (112 and 111) is made in such a mannerthat it can rotate against another by applying a force thereto along thecircumference of the cover (102). In the cover (102), both an ampule(104) containing a medium and another ampule (108) containing adisinfectant are received and held. The inner side of thecover-constituting member (112) is equipped with a protrusion (113)which contacts the ampules (104 and 108). The cover-constituting member(111) is equipped with holes (124 and 128) (the symbols are omitted inthe drawing) which can receive the respective ampules (104 and 108). Acover (102) is constituted in such a manner that the cover-constitutingmember (111) is located on the cover-constituting member (112) (i.e., atthe opposite side of the container (101)) and an ampule (104) is placedin a hole (124) in the cover-constituting member (111) while anotherampule (108) is placed in another hole (128). In the cover-constitutingmember (112), the ampules (104 and 108) placed as such are located so asto encounter to the protrusion (113) as in (a) of FIG. 16.

When, for example, the upper cover-constituting member (111) is rotatedto an extent of 90 degrees in an anticlockwise direction, a tensionwould occur at the contacting part (115) of the ampule with theprotrusion (113) whereupon the ampule (104) is broken and the mediumcontained therein flows out. The medium coming out as such comes into acontainer (101) through one or more perforated holes (not shown) at theengaging portion of the cover (102) with the container (101) by gravityand contacts with the microorganism-collecting end (106 b). When theabove cover-constituting member (111) is further rotated in ananticlockwise direction (totally rotated to an extent of 180 degrees), atension would occur at the contacting part of the ampule (108) (notshown) with the protrusion (113) whereby the ampule (108) is broken andthe disinfectant contained therein flows out. The disinfectantdischarged as such comes into the container (101) by gravity through theperforated hole (not shown) at the engaging portion (joint) of the cover(102) with the container (101) and disinfects/sterilizes the apparatusincluding the incubated microorganism. Alternatively, when the abovecover-constituting member (111) is rotated in a clockwise directioninstead of rotating it to an extent of 180 degrees in an anticlockwisedirection as mentioned above, a tension would occur at a contacting part(116) of the ampule (108) with the protrusion (113) where by the ampule(108) is broken and the disinfectant contained therein flows out.

The microorganism-detecting apparatus of the present invention can bemade (i) in such a structure that a mechanism for breaking thebag-shaped member such as an ampule does not work by being equipped witha pawl or the like so that the bag-shaped member such as the ampule issustained freely from breakage and the contents in the ampule is notreleased until there is a need of releasing the medium or thedisinfectant or (ii) in such a structure that an arrangement is madewherein, unless a certain force is applied, rotation or pushing is notpossible so that the mechanism for breaking the bag-shaped member suchas the ampule does not work. The microorganism-detecting apparatus ofthe present invention may also be in such a manner that a protector suchas a cap is applied to the mechanism for breaking the bag-shaped memberso that the content therein is not released by breakage of thebag-shaped member such as the ampule until there is a need of releasingthe medium or the disinfectant.

FIGS. 17 to 33 illustrate other representative specific examples of themicroorganism-detecting apparatus of the present invention for theirfundamental constitution, particularly the fundamental constitution as astructure for disinfecting and/or sterilizing the cultured medium, etc.after incubation.

FIG. 17 mainly shows the external appearance and shape of themicroorganism-detecting apparatus while its main cross section is shownin FIG. 18.

FIGS. 19 to 33 show each of the elements constituting themicroorganism-detecting apparatus in some more detail. Incidentally, inthose drawings, a microorganism-collecting part or the like is sometimesomitted for better understanding.

A main appearance of a cap member (561) is shown in FIG. 19 while thecross sectional shape of said cap member (561) is mostly shown in FIG.20. FIG. 21 is mostly a cross sectionally perspective view of the lowerarea seen from the section I-I′ of the cap member. An appearance of thecover-constituting member (247) of the microorganism-detecting apparatusis mostly shown in FIG. 22 while the cross sectional view of thecover-constituting member (247) is mostly shown in FIG. 23. FIG. 24mostly shows the appearance of the cover-constituting member (247) fromthe direction of II while FIG. 25 mostly shows the appearance of saidcover-constituting member (247) from the direction of III. FIG. 26mostly shows the appearance of the cover-constituting member (243) ofthe microorganism-detecting apparatus, FIG. 27 mostly shows the crosssectional view of the cover-constituting member (243), FIG. 28 mostlyshows the appearance of the said cover-constituting member (243) fromthe direction IV and FIG. 29 mostly shows the appearance of thecover-constituting member (243) from the direction V, respectively. Theappearance of the container (241) of the micro-organism-detectingapparatus is mostly shown in FIG. 30, and the cross sectional view ofthe container (241) is mostly shown in FIG. 31. FIG. 32 mostly shows theappearance of the container (241) from the direction of VI and FIG. 33mostly shows the cross sectional view of the container (241) at the G-G′section in the microorganism-detecting apparatus equipped with themicroorganism-collecting end (246 b).

In FIGS. 17 and 18, the cover (242) is mainly composed of (a) acover-constituting member (243) which is capable of receiving andholding an ampule (244) containing a medium; (b) a cover-constitutingmember (247) which is capable of receiving and holding an ampule (248)containing a disinfectant; and (c) a cap member (249, 561). A protrusion(252) is installed (formed) at the bottom of the cover-constitutingmember (243), i.e. at the connecting side (259) with the cylindricalcontainer (241). The protrusion (252) may be placed on the entirecircumference of the cover-constituting member (243) whereby thecover-constituting member (243) becomes thick at the connecting part(259) side with the container (241). Alternatively, the protrusion maybe placed (formed) in such a manner that the protrusions are located ina projected manner at some places on the inner side of thecover-constituting member (243), e.g. those in a form of a crosspiece(sash bar). In an example shown in the drawing, it is noted that theprotrusion is installed (formed) having a wedge-shaped cross section atfour sites as given in FIG. 28. Such a shape of the protrusion (252) iseffective in giving a big breaking force at the contacting point A withthe ampule (244). Other examples of the shape, whereby such a functionand merit can be expected, may be suitably selected by anyone skilled inthe art from those which have been widely known in the areas of machinesand instruments, particularly in the area of containers. In a preferredembodiment, a protrusion (297) is formed at the top side of thecover-constituting member (243), i.e. at the cap member (249) sideterminal area, so as to engage with a engaging recess part (299) formedon the cover-constituting member (247) and, when the head part (245) ofthe cover-constituting member (247) is pushed to the direction of thecontainer (241), the bottom (235) of the cover-constituting member (247)can be pushed against the side of the container (241).

In an example shown in this drawing, the protrusion (297) is formed atfour places on the inner wall of a cylindrical member (243), the same asthe protrusion (252) which is shown in FIG. 28 and, corresponding tothat, the connection recess part (299) is formed at four places on theouter wall of the member (247) at the side of the container (241). Thereis no particular limitation for their structures in connection with suchprotrusions (297) and connection recess parts (299) as long as they canengage each other for setting the cover-constituting member (247) at theinner side of the cover-constituting member (243) and thecover-constituting member (247) can slide to the side of the container(241) preferably at the breaking stage of the ampule (244). The shapeand structure can be suitably selected from those which are widely knownin the areas of machines and instruments, particularly in the area ofcontainers, by anyone skilled in the art provided that the function andthe merit as mentioned above can be expected. At the bottom (235) of thecover-constituting member (247), there are one or more through holes(perforated holes) (cf. FIG. 25) so that, when the ampule (248) isbroken, the disinfectant contained therein can flow down into thecontainer (241) as mentioned below. At the bottom side of thecover-constituting member (247), i.e. at the inner ampule (244) side,there is a protrusion (253) by the same manner as in the case of thecover-constituting member (243). Like the above-mentioned protrusion(252), the protrusion (253) may be made on the entire circumference ofthe cover-constituting member (247) whereby the cover-constitutingmember (247) becomes thick at the bottom of the ampule (244) sidethereof. Alternatively, the protrusion (253) may be made in such amanner that the projected parts are partially available at severalplaces on the inner side of the cover-constituting member (247) (such asa protrusion in a form of crosspieces). In an example shown in thisdrawing, it is noted that, as shown in FIG. 24, the protrusion (253) isinstalled in such a manner that it has a wedge-like cross section atfour sites. In a preferred embodiment, threads are made at the top sideof the cover-constituting member (247), i.e. on the end (245) at the capmember (249) side, so as to cooperate with screws (257) made on the capmember (249) and, when the head of the cap member (249) is rotated, thebottom (256) of the cap member (249) can be pushed against the side ofthe ampule (248).

As well noted by referring to FIG. 18 for example, when the head (245)of the cover-constituting member (247) is first pushed down in thedirection of the container (241) and then the cover-constituting member(247) is pushed into the cover-constituting member (243), the bottom(235) of the cover-constituting member (247) pushes down the ampule(244). The pushed-down ampule (244) is then supported by a slope of theprotrusion (252) made on the cover-constituting member (243). As such, atension would occur at the contacting point (A) of the ampule (244) withthe slope of the protrusion (252) as the cover-constituting member (247)is pushed down to the side of the container (241) whereby the lower partof the ampule (244) is broken and the contents therein is allowed torelease. The contents released from the ampule (244) flows into thecontainer (241) by gravity passing the through hole (perforated hole)(cf. FIG. 29) made in the first partition member (296) and contacts withthe microorganism-collecting end (246 b). (Incidentally, in FIG. 28, theampule (244) is omitted and, further, in FIG. 29, themicroorganism-collecting part which is to be made at the central site isomitted.) In an example shown in this drawing, there are four throughholes in a fan-like shape as will be noted by referring to FIGS. 28 and29.

During the disinfecting stage after incubation of the microorganism,when the top (561) of the cap member (249) is rotated and the bottom(256) of the cap member (249) is pushed against the side of the ampule(248), the bottom (256) of the cap member (249) pushes down the ampule(248). The pushed-down ampule (248) is then supported by the slope ofthe protrusion (253) made on the inner side of the cover-constitutingmember (247). As the cap member (249) is rotated and pushed against theside of the container (241), a tension would occur at the contactingpoint (B) of the ampule (248) with the slope of the protrusion (253)whereby the lower part of the ampule (248) is broken and the contentstherein is allowed to release. The contents coming out from the ampule(248) flows into the container (241) by gravity passing through theperforated holes (cf. FIG. 25) made at the bottom (235) of thecover-constituting member (247) and contacts with the incubatedmicroorganism. (Incidentally, in FIG. 24, the ampule (248) is omitted.)In an example shown in this drawing, there are four perforated holes(through holes) in a sectoral form as will be noted by referring toFIGS. 24 and 25. It is preferred in view of convenient use that theampule is made of glass, hard plastics, etc. so that it can be easilybroken upon application of mechanical force (particularly uponapplication of a mechanical force partially).

In the microorganism-detecting apparatus shown in the drawings, theampule containing the medium can be broken by mere sliding and pushingof the cover-constituting member and the cap member (both are screwedand unitedly movable) to supply the medium for incubation to thecontainer while another ampule containing a disinfectant/sterilizer canbe broken to release the disinfectant/sterilizer contained therein intoa container for incubation only when the cap member screw-fitted withthe cover-constituting member accommodating the disinfectant/sterilizerampule is rotated and pushed thereagainst. Therefore, themicroorganism-detecting apparatus is excellent in terms of actual use.

In the microorganism-detecting apparatus of the present invention, it isalso possible that a lock function is installed so that initiation ofthe sliding operation does not take place unless a certain force isapplied thereto. It is further possible that initiation of the rotarymovement of the cap member does not take place unless a certain force isapplied thereto. With regard to a structure giving such a lock function,those which are known in the area of containers may be selected andused. There is no particular limitation for the size of themicroorganism-detecting apparatus but, in terms of portability, thetotal size will be, for example, about 5 to 40 cm in length (preferably,about 10 to 30 cm or, more preferably, about 15 to 25 cm) and about 3 to50 mm in diameter (preferably, about 6 to 30 mm or, more preferably,about 8 to 25 mm). The size of the microorganism-detecting apparatus maybe other than those given hereinabove and freely selected and designedby anyone skilled in the art depending upon the object and operabilityand also by taking the quality, etc. of the material constituting theapparatus into consideration.

FIGS. 34 to 51 illustrate other representative specific examples of themicroorganism-detecting apparatus of the present invention for theirfundamental constitution, particularly the fundamental constitution as astructure for disinfecting and/or sterilizing the cultured medium, etc.after incubation.

FIG. 34 illustrates the assembling of the parts which constitute themicroorganism-detecting apparatus according to the present invention.FIG. 34 shows a cap member (761), an ampule (748) wherein a disinfectantis contained, a cover-constituting member (747) capable of receiving andholding the ampule (748), an ampule (744) containing a medium, acover-constituting member (743) capable of receiving and storing theampule (744), a microorganism-collecting member (746) (comprising arod-shaped element (746 a) and a microorganism-collecting end (746 b))and a container (741). The assembling concept as shown in FIG. 34 isapplicable to the microorganism-detecting apparatus of the presentinvention shown in FIGS. 17 to 33 as well.

FIG. 35 mostly shows the cross-sectional shape of themicroorganism-detecting apparatus. FIG. 36 mostly shows the appearanceof the cover-constituting member (747) of the microorganism-detectingapparatus which is similar to that as shown in FIG. 22. FIG. 37 mostlyshows the cross-sectional view of the cover-constituting member (747)along the line M-M′; FIG. 38 mostly shows the appearance of thecover-constituting member (747) observed from the direction of VII; andFIG. 39 mostly shows the appearance of the cover-constituting member(747) observed from the direction of VIII (In FIGS. 38 and 39, a bridgepart at the end of the cover-constituting member (747) and at thecontainer side, connecting to the protrusion (753), is observed whereinthe thing which is shown as if obliquely crossing to the bridge is a“flash” which is incidentally formed upon molding of the plastic resinand, preferably, such a flash is to be detached in the actual product.)

FIG. 40 mostly shows the cross-sectional shape of the cover-constitutingmember (747) of the microorganism-detecting apparatus similar to thatshown in FIG. 23 wherein (778) is a small convex ridge formed on theinner wall of the cover-constituting member (747) while (753) is aprotrusion for breaking the ampule (748). Unlike the (253) shown in FIG.23 (and also in FIG. 24), this (753) is formed only at one place (cf.FIG. 38) and, in addition, the shape is different (cf. FIG. 40). In themicroorganism-detecting apparatus, such an ampule-breaking function anda structure for enabling this function are also characteristic featuresof the present invention. Accordingly, an apparatus and materials(members) having such a function constitute a part of the presentinvention.

As will be noted from FIG. 37, there is a concave () and shallow groove(777) in this cover-constituting member (747). The groove (777) isformed in such a manner that, as will be illustrated later, it can beengaged with a small convex ridge (781, 782, 783, etc.) formed on theinner wall of the cover-constituting member (743) of themicroorganism-detecting apparatus whose cross-sectional shape is mostlyshown by FIG. 44. Although, in an example as shown in the drawing,length of all of four grooves (777) is the same, numbers and length ofthe groove may be suitably changed upon necessity. In FIG. 40, noperforated hole is illustrated through which a disinfectant (liquid)contained in the ampule (748) flows down by gravity when the ampule(748) is broken, but such a perforated hole (through hole) can be easilyunderstood by referring to FIGS. 38 and 39.

In FIGS. 35 and 40, it is shown that the inner diameter of the aboutone-third portion of the cover-constituting member (747) at the side ofthe container (741) is either broader than or the same as the innerdiameter at the side of its cap member (761), although it is alsopreferred that the inner diameter is narrower than that at the side ofits cap member (761) so that the movement of the receiving ampule (748)is restricted.

FIG. 41 mostly shows the appearance of the cover-constituting member(743) of the microorganism-detecting apparatus which is the same as thatas shown in FIG. 26. FIG. 42 mostly shows the appearance of thecover-constituting member (743) observed from the direction of IX; andFIG. 43 mostly shows the appearance of the cover-constituting member(743) from the direction of X. By referring to FIGS. 35, 42 and 43, thepresence of the through holes (perforated holes) through which themedium (liquid) contained in the ampule (744) flows down by gravity canbe easily understood. In this example, there are three perforated holes(through holes) in a shape of a fan. When the ampule (748) is broken,the disinfectant (liquid) contained in the ampule (748) flows down bygravity through the perforated holes and comes into the container (741).

There are small convex ridges (781, 782, 783 and 784) on the inner wallof the cover-constituting member (743). In FIG. 44, there are two smallconvex ridges (781 and 783) in the plane N-N′ (cf. FIG. 45 whichillustrates a cross section along the plane N-N′) but, in the P-P′plane, there are four (781, 782, 783 and 784) (cf. FIG. 46 whichillustrates a cross section along the plane P-P′). FIG. 47 shows a crosssection along the plane Q-Q′. The small convex ridge (781) rangesconsecutively to (785) and further extends to (752) while, among otherssuch as (782), it ranges to (786) but has no protrusion such as (752).Unlike (252) shown in FIG. 27, there is only one (752) in the presentmicroorganism-detecting apparatus and its shape is different as well.

In the microorganism-detecting apparatus of the present invention, suchan ampule-breaking function and a structure which makes it possible arealso the characteristic features of the present invention and apparatusand materials (members) having such functions also constitute a part ofthe present invention.

FIG. 48 shows the oblique outer view of the cover-constituting member(743) made up of a transparent (or translucent) material in a partlyperspective manner. In the drawing, the arrangement of small convexridges (781, 782 and also 785 and 752 ranging therefrom) formed on theinner wall of the cover-constituting member (743) is visible andunderstandable. (781) extends to the cover-constituting member(747)-receiving side longer than (782). The small convex ridges may bemade in such a manner that the convex is made larger (i.e. projected toa higher extent) from the place (785) ranging therefrom for allowing thereceived ampule (744) to be loosely fixed. The area (790) at the outerwall of the cover-constituting member (743) is made hardly slippery byforming a finely indented surface, like a frosted glass. Such a finelyindented surface, like a frosted glass, may be at either a part of orwhole of the cover-constituting member (743).

The basic shape of the apparatus shown in FIGS. 34 to 51 is the same asthat of the microorganism-detecting apparatus of the present inventionshown in FIGS. 17 to 33 but its characteristic feature is as follows:

Until an initiation of incubation, the cover-constituting member (747)is set free by means of a locking mechanism from pushing into thecover-constituting member (743) to break the ampule (744) received inthe cover-constituting member (743), while, upon the initiation ofincubation, the engagement is easily unlocked by merely rotating thecover-constituting member (747) against the cover-constituting member(743) whereby the cover-constituting member (747) can be pushed into thecover-constituting member (743) so that the ampule (744) is broken andthe medium can be supplied to the container (741).

FIGS. 49 to 51 illustrate the conceptual outlines of the lockingmechanism and unlocking thereof. At the state prior to use of themicroorganism-detecting apparatus, when the cover-constituting member(747) is inserted into the cover-constituting member (743) in such amanner that (i) a long and convex () small ridge (781) formed on theinner side of the cover-constituting member (743) and (ii) a concave ()and shallow groove (777) formed on the inner side of thecover-constituting member (747) are not engaged each other, both thecover-constituting members are fixed due to a big frictional engagingforce between the convex small ridge (781) and the outer surface of themember (747). As a result, each member stays nearly at the position asshown by FIG. 49. In other words, the fixation is in such a manner thatthe member (747) cannot be pushed into the member (743) (i.e. againstthe side of the container) beyond the stopping position.

When, upon breaking the ampule (744), for example, thecover-constituting member (747) is rotated to the direction as shown byan arrow in FIG. 49, (i) the convex small ridge (781) and (ii) theconcave and shallow groove (777) are engaged with each other as shown inFIG. 50, so that the frictional engaging force generated between theconvex small ridge (781) and the outer surface of the cover-constitutingmember (747) disappears whereupon the cover-constituting member (747)can now be pushed in the direction further as shown by an arrow in FIG.50. Then, when the cover-constituting member (747) is inserted as shownin FIG. 51, the bottom of the cover-constituting member (747) pushes thehead of the ampule (744) whereupon the ampule is pushed to theprotrusion (752) followed by breakage. In this example, (i) the convexridges (four in total) formed on the inner side of thecover-constituting member (743) and (ii) the concave shallow grooves(four in total) (recessing parts) formed at the outer side of thecover-constituting member (747) are engaged with each other at the end.Simultaneously, the engaging surface of the cover-constituting member(743) and that of the cover-constituting member (747) are tightlyengaged each other at the side of the cap member (761). Thus, the innerwall of the cover-constituting member (743) and the outer wall of thecover-constituting member (747) are tightly contacted with each other,thereby achieving a tight sealing to some extent. In themicroorganism-detecting apparatus, such a locking mechanism and astructure for making it possible or available are a part of thecharacteristic features of the present invention and the apparatushaving such a mechanism (or function) and materials (members) thereofconstitute a part of the present invention.

The microorganism-detecting apparatus of the present invention as shownin FIGS. 34 to 51 are the same as those shown in FIGS. 17 to 33 in termsof basic shape, size, etc. and, accordingly, details of other materialswill be and is to be understood similarly.

FIG. 52 partly shows a partial cross sectional view of the connectingpart (776 a) of the cover-constituting member (743) with the container(741). It is understood that an end of the microorganism-collectingmember (746) can be inserted into and fixed with the inlet (789) for themicroorganism-collecting part (member). At the basement of the inlet(789), there is one or more perforating holes for communicating a mediumand a disinfectant. As will be noted by referring to FIG. 52, aprotrusion (789 a) is formed in a manner surrounding the inlet (789).When an end of the container (741) is screwed into a connecting part(776 a) of the cover-constituting member (743), the protrusion (789 a)assists a close engagement (connection) of the cover-constituting member(743) with the container (741). In other words, when a plastic resinsuch as polypropylene is used as a constituting material for theengaging portions connecting between the cover-constituting member (743)and the container (741), it is possible to tightly seal it throughutilizing the elasticity of the material of the members. In themicroorganism-detecting apparatus according to the present invention,the structure of such a engaging part (776 a) of the cover-constitutingmember (743) with the container (741) is also one of the characteristicfeatures of the present invention and the apparatus having such afunction and materials thereof also constitute a part of the presentinvention.

FIGS. 53 to 69 illustrate other representative specific embodiments ofthe microorganism-detecting apparatus according to the present inventionfor their fundamental constitution, particularly the fundamentalarrangements suitable for disinfecting and/or sterilizing the culturedmedium, etc. after incubation.

In these drawings, the microorganism-detecting apparatus according tothe present invention is shown which has a mechanism for allowing aperson to keep and carry it securely and which is designed in a mannerthat the person can avoid the breakage of (i) an ampule containing adisinfectant and (ii) an ampule containing a medium.

FIGS. 53, 55, 57, 59, 61, and 63 show a perspective view of themicroorganism-detecting apparatus according to the present invention,respectively. FIG. 54 illustrates the assembling of the parts 1245 (and1561) and 1243 for the microorganism-detecting apparatus of FIG. 53.FIG. 56 illustrates the assembling of the parts 1245 (and 1561) and 1243for the microorganism-detecting apparatus of FIG. 55. FIG. 58illustrates the assembling of the parts 1561 and 1245 for themicroorganism-detecting apparatus of FIG. 57. FIG. 60 illustrates theassembling of the parts 1561 and 1245 for the microorganism-detectingapparatus of FIG. 59. FIG. 62 illustrates the assembling of the parts1561, 1245 and 1243 for the microorganism-detecting apparatus of FIG.61. FIG. 64 illustrates the assembling of the parts 1561, 1245 and 1243for the microorganism-detecting apparatus of FIG. 63.

In FIGS. 53 and 57, a guide groove (guide space) (1004, 1007) is formedat two sites while the guide groove (guide space) is formed at one sitein FIGS. 55 and 59. In FIG. 61, the member (1243) has two guide grooves(guide spaces) (1004); the member (1245) has two stoppers (1005) and twoguide grooves (guide spaces) (1007); and the cap member (1561) has twostoppers (1008)). In FIG. 63, the member (1243) has one guide groove(guide space) (1004); the member (1245) has one stopper (1005) and oneguide groove (guide space) (1007); and the cap member (1561) has onestopper (1008). One or optionally more guide grooves (guide spaces) canbe formed in each member. Although, in the drawings, the guide portion(1004) is formed through the side wall of the member (1243), it ispossible to form a shallow guide groove by reducing the inner side wallof the member (1243) so as to make it partially thin (by cutting theinner side wall thereof) without a through hole. In the drawings, theguide (1004) is formed in the side wall of the member (1243) and astopper (1005) is formed on the side wall of the member (1245) at theengaging side with the member (1243). Alternatively, the guide (1004)may be formed in the side wall of the member (1245) and a stopper (1005)may also be formed on the side wall of the member (1243). Similarly, aguide portion (1007) and a stopper (1008) can be formed in the members.The shape of the guide groove (guide space) is not limited to butincludes a L shape, those as illustrated in FIG. 70 and the like.

FIG. 65 shows an appearance of the members (1561, 1245) in anotherrepresentative embodiment of the microorganism-detecting apparatusaccording to the present invention. FIG. 66 shows an appearance of themembers (1561, 1245) in another representative embodiment of themicroorganism-detecting apparatus according to the present invention.FIG. 67 shows the member 1243 capable of engaging with the members (1245(and 1561)) illustrated in FIG. 65. FIG. 68 is a schematic diagram forexplaining the relationship between the guide portion (1004) and thestopper (1005). FIG. 69 is an enlarged partial view in connection withthe relationship between the guide portion (1004) and the stopper(1005).

Prior to use of the microorganism-detecting apparatus according to thepresent invention, the member (1245) is engaged with the member (1243)(FIG. 54) and the stopper (1005) is positioned at the upper side of theguide (1004) (FIG. 53). Referring to FIG. 68, the stopper (1005) islocated at the site B in FIG. 68-1.

Upon breaking the ampule (upon use thereof), the stopper (1005) is movedbeyond a pawl portion (neck) (A) (FIG. 68-2 and FIG. 69) by turning themember (1245) whereby the member (1245) can be pushed into the member(1243). Thus, the stopper (1005) can be moved to the position (D) (InFIG. 53, it corresponds to the stopper (1005′)). If necessary, a pawlportion (neck) (C) may be formed in order to fix the stopper (1005)(FIG. 68-3). In FIGS. 55 to 64, each apparatus has the same mechanismand action.

FIG. 65 shows an embodiment of the microorganism-detecting apparatusaccording to the present invention wherein a measure for securing theadhesion (sealing) between the member (1243) and the member (1245) isset at the surface region (1351) on the outer wall of the member (1245).The adhesion (sealing) may be achieved by various techniques includingfrosting of a surface, coating with rubber, etc. and the usefultechnique can be selected from those known to a person skilled in theart such as the field of container. Although FIG. 65 also shows thepossibility that a measure for securing the adhesion between the member(1243) and the member (1245) will be made at the surface region (1350)on the outer wall of the member (1245), this measure is generallydisposed at the surface region (1351) when the stopper (1005) -guide(1004) system is formed.

FIG. 67 shows an embodiment of the microorganism detecting apparatusaccording to the present invention wherein a measure for securing theadhesion between the members (deeply shadowed area) is installed on theinner wall of the member (1243) which corresponds to the surface region(1351) formed on the member (1245). Similarly to FIG. 65, FIG. 67 alsoshow the possibility that a measure for securing the adhesion will bemade on the slantingly lined area. In FIG. 67, a shallow groove (1360)is formed so as to facilitate an insertion of the stopper (1005) intothe guide portion (1004).

In FIG. 66, the stopper (1352) has a structure capable of being flexiblypressed down against the outer wall side of the member (1245) uponinsertion of the member (1245) into the member (1243). The stopper(1352) is movable inwardly and outwardly by a deflecting force. Inaddition, the mechanism required in keeping and carrying the apparatuswithout the breakage of (a) the ampule for a medium and (b) the ampulefor a disinfectant may include an engagement of a convex portion formedon one member with a concave portion formed on another member for (i)the members (1245) and (1243) and for (ii) the members (1561) and(1245), a combination of a stopper removable from the detectingapparatus therewith. The mechanism may comprise a mark attached thereto,which indicates a position for fixing each member.

In accordance with the present invention, it has been found that it ispossible to measure how many viable microorganism cells are presentprior to an initiation of incubation, substantially in a closed system,such as the microorganism-detecting apparatus of the present invention.Utilization of the microorganism-detecting apparatus of the presentinvention enables a person to easily, securely and portably estimate thenumber of viable microorganism cells present in foods, cooking tools andthe like. Accordingly, it is possible to estimate a degree of pollutionquickly for various samples including cooking utensils, e.g. kitchenknife and chopping board, foods, and beverages or surroundings, by usingthe microorganism-detecting apparatus of the present invention. Throughthe use of the apparatus of the present invention, a person can easily,conveniently, securely and safely judge what kind or species ofmicroorganism it is and/or determine the amount of microorganismspresent.

The measurement of viable cells can be carried out by culturing the cellin a liquid medium contained in the apparatus of the present inventionfollowed by measuring absorbance, turbidity, etc. for the medium withouttaking it out of the apparatus. The measurement may be carried out by ameasuring instrument or machine, by visual observation including acomparison with a symbol mark and the like, or by a combination thereof.The visual observation may include a technique employing labels such ascharacters, lines, pictures and dots wherein a recognition degree isestimated. The label for recognition may be attached to or marked(painted) on a swab (microorganism-collecting member), an inner wall ofthe container receiving the medium for culturing cells, or an outer wallthereof. The label for recognition may be suitably selected in view ofits shape, color, depth and lightness. For the quantitative measurement,it is preferred to avoid a pigment in a medium for culturingmicroorganisms. In the present measurement, it is possible to estimatethe number of cells present at a starting point.

Incidentally, in FIG. 1 and in FIGS. 3 to 70, illustrations are made forconcentrating an the characteristic points of themicroorganism-detecting apparatus according to the present invention. Itis to be understood that some of the details may be omitted there.Consequently, it is to be understood that the apparatus having thecharacteristic features of the apparatus according to the presentinvention shown in this specification, drawings, and the attached claimsincluding those wherein various modifications are made and each of theconstituting parts or members is connected each other in more detailedmanner are also included in the coverage of the present invention.

Merit of the Invention

As mentioned hereinabove, the present invention provides a selectivemicroorganism-detecting apparatus where certain microorganisms(particularly pathogenic ones and, more particularly, those for foodpoisoning) can be easily, securely and simply detected and/oridentified. The apparatus can be expected to be stored for a long periodand is in such a shape (arrangement) that it can be immediately used atany time when necessary and that it can be easily and convenientlycarried. Thus, the present invention provides a selectivemicroorganism-detecting apparatus which is especially suitable forprivate and domestic use where toxic microorganisms such as those forfood poisoning can be easily, securely and simply detected/identifiedand, even after use, is able to be disposed safely, surely and easily.

The microorganism-detecting apparatus of the present invention can bevery easily used privately and domestically and, therefore, it is nowpossible to detect the microorganism and also to establish a preventingmeasure against food poisoning by a self-active basis not performed byprofessionals such as those in public health centers. After incubationof the microorganism, the microorganism-detecting apparatus of thepresent invention is not made into an open system, for instance, byopening a cover. Even if a pathogenic microorganism is containedtherein, therefore, it is easy to change the used apparatus to adisposable form and then to discard it. Moreover, there is no need thatthe apparatus used for incubation is opened and disinfected by adding adisinfectant to the microorganism-containing culture liquid with a toolsuch as a pipette. Accordingly, troublesome operations can be eliminatedand there is no danger. No professional technique, skillfulness andapparatus is necessary to operate the apparatus of the present inventionfor detecting microorganisms. Further, in the microorganism-detectingapparatus of the present invention, it is unnecessary to prepare adisinfectant solution at all times separately from the container forincubation. Therefore, the apparatus is quite good in terms ofportability and easy handling.

EXAMPLES

Described below are examples of the present invention which are providedonly for illustrative purposes, and not to limit the scope of thepresent invention. In light of the present disclosure, it should beunderstood that numerous embodiments within the scope of the claims willbe apparent to those of ordinary skill in the art.

Example 1

(Apparatus for Detection of Staphylococcus aureus and PreparationThereof)

By referring to FIG. 5, the following materials were used to constructthe microorganism-detecting apparatus having the following size:

Container (1): a hollow cylinder having a bottom (inner diameter: 8.5mm; height: 150 mm) made of polyethylene (thickness: 0.6 mm)

Cover (2): a hollow cylinder having a bottom (inner diameter: 10 mm;length: 53 mm) made of polyethylene (thickness: 0.6 mm)

First bag-shaped member (4): a hollow cylinder having a bottom (innerdiameter: 7.5 mm; length: 40 mm) made of glass (thickness: 0.5 mm)

Partition member (5): a disk-shaped body with a diameter of 6 mm made ofpolyethylene (thickness: 1 mm)

Rod-shaped element (6 a): made of polyethylene; diameter: 2 mm; length:135 mm

Microorganism-collecting end (6 b): an end of “swab”, made of absorbentcotton (diameter: 5 mm; length: 15 mm)

Disk member (7): filter paper (thickness: 1 mm; diameter: 6 mm)

The antibiotic substances as shown in Table 1 were impregnated in thedisk member (7), located between the bottom of the container (1) and themicroorganism-collecting end (6 b) in such amounts that concentrationsof 10 μg/ml of aztreonam, 8 μg/ml of polymyxin B and 5 μg/ml offluconazole were obtained upon unification with the medium.

Medium (3): a medium having the composition as shown in Table 1 (exceptthe antibiotics).

TABLE 1 A) Screening Medium for Detection of Staphylococcus aureus(Modified Mannitol-Salt Medium) Myosate Peptone 2.5 g PolypeptonePeptone 10.0 g Yeast Extract 2.5 g D-Mannitol 10.0 g Lithium Chloride5.0 g Sodium Chloride 40.0 g Phenol Red 25 mg Distilled Water 1000 ml pH7.5 Sterilized by autoclaving at 115° C. for 15 minutes. Secondary Test:Disk for detection of phosphatase Disk: Aztreonam 1 to 15 μg/mlPolymyxin B 1 to 15 μg/ml Fluconazole 1 to 10 μg/ml

After preparing a detecting apparatus for Staphylococcus aureus havingthe above composition, it was sterilized by irradiating with gamma-rayat 1 to 30° C. for one minute. The apparatus after the sterilization wasstored in a bag made of polyethylene film (thickness: 100 μm) laminatedwith (vaporized) aluminum for shielding the light until immediatelybefore use.

Example 2

(Detecting Apparatus for Vibrio parahaemolyticus and PreparationThereof)

A detecting apparatus for detecting Vibrio parahaemolyticus wasprepared, sterilized and stored by the same manner as in Example 1 withan exception that composition for the medium (3) and antibiotics werechanged as shown below (Table 2) by referring to FIG. 5. At that time,the antibiotic substances as shown in Table 2 were impregnated in a diskmaterial (7) in such amounts that concentrations of 10 μg/ml ofpolymyxin B, 5 μg/ml of fluconazole and 5 μg/ml of potassium telluritewere obtained upon unification with the medium.

TABLE 2 B) Screening Medium for Detection of Vibrio parahaemolyticus(Modified Salt-Polymyxin Medium) Polypeptone Peptone 10.0 g YeastExtract 5.0 g Sodium Chloride 20.0 g Saccharose 15 g Sodium dodecylsulfate 1.0 ml Bromthymol Blue 40.0 mg Cresol Red 40.0 mg DistilledWater 1000 ml pH 7.2 Sterilized by autoclaving at 115° C. for 15minutes. Secondary Test: Disk for detection of cytochrome oxidase Disk:Polymyxin B 1 to 15 μg/ml Fluconazole 1 to 10 μg/ml Potassium tellurite1 to 10 μg/ml

Example 3

(Detecting Apparatus for Salmonella and Preparation Thereof)

A detecting apparatus for detecting Salmonella was prepared, sterilizedand stored by the same manner as in Example 1 with an exception thatcomposition for the medium (3) and antibiotics were changed as shownbelow (Table 3) by referring to FIG. 5. At that time, the antibioticsubstances as shown in Table 3 were impregnated in a disk member (7) insuch amounts that concentration of 5 μg/ml of fluconazole was obtainedupon unification with the medium.

TABLE 3 C) Screening Medium for Detection of Salmonella (ModifiedXylose-Lysine Medium) Yeast Extract 5.0 g Sodium Chloride 5.0 gSaccharose 5.0 g Lactose 5.0 g Xylose 4.0 g Lysine 10.0 g Sodium dodecylsulfate 1.0 ml 0.2% Bromocresol Purple 12.0 ml Ammonium Iron Citrate 0.5g Sodium Thiosulfate Pentahydrate 0.2 g Distilled Water 1000 ml pH 6.8Sterilized by autoclaving at 115° C. for 15 minutes. Disk: Fluconazole 1to 10 μg/ml

Example 4

(Confirmation of Selectivity of a Detecting Medium for Staphylococcusaureus)

Selectivity of the medium for detecting Staphylococcus aureus preparedin Example 1 was confirmed to be as follows:

Twenty-two kinds of microorganisms as shown in the following Table 4were dispersed in 1.5 ml of the medium for detecting Staphylococcusaureus prepared in Example 1 to make the concentration about 10⁶cells/ml at the initiation of the incubation, cultured in air at 37° C.for 24, 48 or 72 hours and, out of the color change (if any) of PhenolRed (a pH indicator contained in the above medium), a judgment was madewhether the growth of the microorganism was noted. When the color toneof the medium after the incubation turned yellow or red, the conclusionwas done as “positive (+)” (growth of the microorganism was noted) or“negative (−)” (growth of the microorganism was not noted),respectively.

TABLE 4 A) Modified Mannitol Salt Base Medium (Grain-Positive Cocci andGrain-Negative Cocci) Gram Positive/ S. aureus S. aureus S. aureus Sagal- S. pyo- S. epider- S. epider- S. sapro- S. haemo- E. faeca- E.faeca- Antibiotics 209P TEN 003 actiae genes midis 2 midis 5 phyticuslyticus lis 18 lis 7862 Aztreonam 24 hr + + + − − − − + + − − 10 μg/ml +Polymyxin B 48 hr + + + − − − − + + − − 8 μg/ml + Fluconazole 72hr + + + − − − − + + − + 5 μg/ml Gram Negative/ Salmo- Entero- Escher-Kleb- Proteus Proteus Proteus Pseudo- Pseudo- Antibiotics nella Serratiabacter Shigella ichia siella 1 19 20 monas 1 monas 2 Aztreonam 24 hr − −− − − − − − − − − 10 μg/ml + Polymyxin B 48 hr − − − − − − − − − − − 8μg/ml + Fluconazole 72 hr − − − − − − − − − − − 5 μg/ml Positive (+):Color of the medium turned yellow; Negative (−): Color of the mediumturned red

The result is as shown in Table 4. It is clear from the result of Table4 that, when the medium for detecting Staphylococcus aureus of thepresent invention was used, proliferation of Gram-negative microorganismand Eumycetes was effectively inhibited by antibiotic and antifungalsubstances adsorbed with said medium (3) and said disk (7).

Among the microorganisms tested hereinabove, S. epidermidis did notdecompose mannitol contained in the medium (3) and, therefore, there wasno change in the color tone of the medium.

According to the experiments conducted by the present inventors, it wasfound that there were some microorganisms other than Staphylococcusaureus which were “positive” in this medium and that they werecoagulase-negative Staphylococcus (CNS), E. faecalis, E. faecium, etc.However, those microorganisms were able to be discriminated fromStaphylococcus aureus by a phosphatase test or by a coagulase test (cf.“Directions for Hygienic Tests: Staphylococcus”).

In a phosphatase test, incubation was conducted by the above-mentionedmedium using a disk for detection of phosphatase (filter paper adsorbedwith 4-methylumbelliferyl phosphoric acid) and, after that, fluorescenceof 4-methylumbelliferone was detected by irradiating with ultravioletray (366 nm) whereby its presence was able to be confirmed.

Example 5

(Confirmation of Selectivity of a Detecting Medium for Vibrioparahaemolyticus)

Selectivity of the medium for detecting Vibrio parahaemolyticus preparedin Example 2 was confirmed to be as follows:

Twenty-two kinds of microorganisms as shown in the following Table 5were dispersed in 1.5 ml of the medium for detecting Vibrioparahaemolyticus prepared in Example 2 to make the concentration about10⁶ cells/ml at the initiation of the incubation, cultured in air at 37°C. for 24, 48 or 72 hours and, out of the color change (if any) ofBromthymol Blue and Cresol Red (pH indicators contained in the abovemedium), a judgment was made whether the growth of the microorganism wasnoted. When the color tone of the medium after the incubation turnedyellow or green, the conclusion was done as “positive (+)” (growth ofthe microorganism was noted) or “negative (−)” (growth of themicroorganism was not noted), respectively.

TABLE 5 B) Modified Polymyxin Salt Base Medium (Gram-Positive Cocci andGram-Negative Cocci) Gram Positive/ S. aureus S. aureus S. aureus Sagal- S. pyo- S. epider- S. epider- S. sapro- S. haemo- E. faeca- E.faeca- Antibiotics 209P TEN 003 actiae genes midis 2 midis 5 phyticuslyticus lis 18 lis 7862 Polymyxin B 24 hr − − − − − − − − − − − 10μg/ml + Fluconazole 48 hr − − − − − − − − − − − 5 μg/ml + Potassium 72hr − − − − − − − − − − − Tellurite Gram Negative/ Salmo- Entero- Escher-Kleb- Proteus Proteus Proteus Pseudo- Pseudo- Antibiotics nella Serratiabacter Shigella ichia siella 1 19 20 monas 1 monas 2 Polymyxin B 24 hr −− − − − − − + + − − 10 μg/ml + Fluconazole 48 hr − − − − − − + + + − − 5μg/ml + Potassium 72 hr − − − − − − + + + − − Tellurite Positive (+):Color of the medium turned yellow; Negative (−): Color of the mediumturned green

The result is as shown in Table 5. It is clear from the result of Table5 that, when the medium for detecting Vibrio parahaemolyticus of thepresent invention was used, growth of Gram-positive microorganisms wasinhibited by antibiotic substances while that of Gram-negativemicroorganism and most of Eumycetes were effectively inhibited byantibiotic substances (polymyxin B, fluconazole and potassium tellurite)adsorbed with said disk (7).

According to the experiments conducted by the present inventors, it wasfound that there were some microorganisms other than Vibrioparahaemolyticus which were “positive” in this medium and that they wereV. cholerae, V. vulnificus, Proteus spp., etc. However, thosemicroorganisms were able to be discriminated from Vibrioparahaemolyticus by an oxidase test (a cytochrome oxidase reaction; cf.Kovacs: Nature (London), 178, 703 (1956)).

Example 6

(Confirmation of Selectivity of a Detecting Medium for Salmonella)

Selectivity of the medium for detecting Salmonella prepared in Example 3was confirmed to be as follows:

Twenty-two kinds of microorganisms as shown in the following Table 6were dispersed in 1.5 ml of the medium for detecting Salmonella preparedin Example 3 to make the concentration about 10⁶ cells/ml at theinitiation of the incubation, cultured in air at 37° C. for 24, 48 or 72hours and, out of the color change (if any) of Bromcresol Purple (a pHindicator contained in the above medium), a judgment was made whetherthe growth of the microorganism was noted. When the color tone of themedium after the incubation turned “black” or “purple or yellow”, theconclusion was done as “positive (+)” (growth of the microorganism wasnoted) or “negative (−)” (growth of the microorganism was not noted),respectively. Hydrogen sulfide-producing microorganisms such asSalmonella changed the color of the medium to black by the reaction ofthiosulfate contained in the medium with ammonium iron citrate.

TABLE 6 C) Modified Xylose-Lysine Base Medium (Grain-Positive Cocci andGram-Negative Cocci) Gram Positive/ S. aureus S. aureus S. aureus Sagal- S. pyo- S. epider- S. epider- S. sapro- S. haemo- E. faeca- E.faeca- Antibiotics 209P TEN 003 actiae genes midis 2 midis 5 phyticuslyticus lis 18 lis 7862 Fluconazole 24 hr − − − − − − − − − − − 5 μg/ml48 hr − − − − − − − − − − − 72 hr − − − − − − − − − − − Gram Negative/Salmo- Entero- Escher- Kleb- Proteus Proteus Proteus Pseudo- Pseudo-Antibiotics nella Serratia bacter Shigella ichia siella 1 19 20 monas 1monas 2 Fluconazole 24 hr + − − − − − − − − − − 5 μg/ml 48 hr + − − − −− − − − − − 72 hr + − − − − − + − − − − Escherichia coli Citrobacter sppKiebsiella spp color of the medium: yellow Enterobacter spp Proteus sppShigella spp Serratia spp color of the medium: purple Pseudomonas sppPositive (+): Color of the medium turned yellow; Negative (−): Color ofthe medium turned purple or yellow Salmonella . . . color of the medium:black

The result is as shown in Table 6. It is clear from the result of Table6 that, when the medium for detecting Salmonella of the presentinvention was used, growth of Gram-positive microorganisms was inhibitedby the antibiotic substances while that of Eumycetes was effectivelyinhibited by the antibiotic substances (fluconazole) adsorbed with saiddisk (7).

According to the experiments conducted by the present inventors, it wasfound that there were some Gram-negative microorganisms changed thecolor tone of this medium from purple to yellow. Examples of such acolor change were as follows:

Microorganism which changed the color to black color:

Salmonella;

Microorganisms which changed the color to yellow:

Escherichia coli, Citrobacter spp., Klebsiella spp.,

Enterobacter spp. and Proteus spp.; and

Microorganisms which changed the color to purple:

Shigella spp., Serratia spp. and Pseudomonas spp.

Example 7

(Use of the Apparatus for Detecting Staphylococcus aureus)

A bag for the apparatus for detecting Staphylococcus aureus prepared inExample 1 was broken to take out said apparatus, then a cover (2) (and amicroorganism-collecting part (6)) were taken out from the apparatus(cf. FIG. 2(a)) and the microorganism-collecting end (6 b) was rubbedfor several times against the surface of a cooking device (such as achopping board) (cf. FIG. 2(b)).

After that, the above cover (2) was inserted into the apparatus again,the microorganism-collecting end (6 b) was kept at a predeterminedposition in the container, the first bag-shaped member (4) was broken bystrongly compressing from outside of the cover (2) using a compressingdevice (“a breaking device” in a broad clip-like shape) (not shown) andthe medium (3) enclosed in said first bag-shaped member (4) was made tofall down into the container. As a result, the microorganism-collectingend (6 b) in the the microorganism-collecting part (6) dipped into themedium (3) (it was possible to prevent the stuffs other than the medium(such as broken pieces of the first bag-shaped member (4)) fromfalling-down into the container).

Incubation was then conducted at 37° C. for 16 to 24 hours in such astate that the microorganism-collecting end (6 b) of themicroorganism-collecting part (6) was dipped in a medium (3) whereuponthe color tone of the medium (3) changed from red to yellow in some ofthe cooking device (chopping board). Thus, the presence ofStaphylococcus aureus was confirmed. The cooking device where thepresence of Staphylococcus aureus was confirmed as such was wiped forseveral times with a gauze moisturized with a 0.2 to 0.5% aqueouschlorohexidine solution (“Hibiden” (trade name); manufactured bySUMITOMO PHARMACEUTICALS CO. LTD., Japan) followed by washing for tenminutes with tap water. The cooking device (chopping board) which wastreated by cleaning with invert soap and washing with water as such wassubjected to a test for detecting Staphylococcus aureus as same as abovewhereupon the color tone of the medium (3) was unchanged but was stillin yellow.

This meant that no Staphylococcus aureus was detected. Thus, it wasconfirmed that Staphylococcus aureus could be effectively removed by theabove-mentioned disinfecting treatment.

(Disinfecting/Sterilizing Treatment in the Used Apparatus for DetectingStaphylococcus aureus)

The second bag-shaped member (8) was broken by strongly compressing itscover (2) from outside using a compressing device (a “breaking device”in a broad clip-shaped one) (not shown) whereby adisinfectant/sterilizer (9) (a 5 to 10% aqueous solution ofchlorohexidine (trade name: “Hibiden”; manufactured by SUMITOMOPHARMACEUTICALS CO. LTD., Japan)) enclosed in said second bag-shapedmember (8) was released down into the container. As a result, it is nowpossible that a disinfecting/sterilizing treatment of the inner side ofthe container (1) including the medium is conducted without detachingthe cover (2) from the container (1).

Example 8

(Use of Apparatus for Detecting Vibrio parahaemolyticus and Salmonella)

The same operation as in Example 7 was conducted except that theapparatuses for detecting Vibrio parahaemolyticus and for Salmonellaprepared in Examples 2 and 3, respectively, were used to confirm thepresence of food poisoning microorganism in cooking devices whereupon,in some cooking devices, presence of Vibrio parahaemolyticus orSalmonella was confirmed.

When those cooking devices were wiped with invert soap and washed withwater by the same manner as in Example 7, the presence of Vibrioparahaemolyticus and Salmonella was not noted after said disinfectingtreatment. Thus, it was now confirmed that Vibrio parahaemolyticus orSalmonella could be effectively removed by the above-mentioneddisinfecting treatment.

(Disinfecting/Sterilizing Treatment of Used Apparatus for DetectingVibrio parahaemolyticus and Salmonella)

The second bag-shaped member (8) was broken by strongly compressing itscover (2) from outside using a compressing device (a “breaking device”in a broad clip-shaped one) (not shown) whereby adisinfectant/sterilizer (9) (a 5 to 10% aqueous solution ofchlorohexidine (trade name: “Hibiden”; manufactured by SUMITOMOPHARMACEUTICALS CO. LTD., Japan)) enclosed in said second bag-shapedmember (8) was released down into the container. As a result, it is nowpossible that a disinfecting/sterilizing treatment of inner side of thecontainer (1) including the medium is conducted without detaching thecover (2) from the container (1).

Example 9

The same operation as in Example 7 was conducted for confirming thepresence of food poisoning microorganism in cooking devices except thatthe detecting apparatuses having basic structures as shown in FIGS. 12to 16 were used and that the screening media for Staphylococcus aureus,for Vibrio parahaemolyticus and for Salmonella mentioned in Examples 1,2 and 3, respectively, and the antibiotic substances mentioned thereinwere jointly used whereupon, in some cooking devices, presence ofStaphylococcus aureus, Vibrio parahaemolyticus or Salmonella wasconfirmed.

When those cooking devices were wiped with invert soap and washed withwater by the same manner as in Example 7, the presence of Staphylococcusaureus, Vibrio parahaemolyticus and Salmonella was not noted after saiddisinfecting treatment. Thus, it was now confirmed that Staphylococcusaureus, Vibrio parahaemolyticus or Salmonella could be effectivelyremoved by the above-mentioned disinfecting treatment.

(Disinfecting/Sterilizing Treatment of the Used Detecting Apparatus)

The second bag-shaped members (48, 64 (or 68), 88, 108) were broken bystrongly compressing their covers (42, 62, 82, 102) from outside using abreaking function of the apparatus as shown in FIGS. 12 to 16 (such as49, 461, 56, 53; 73, 74, 77; 93; 113) whereby a disinfectant/sterilizer(9) (a 5 to 10% aqueous solution of chlorohexidine (trade name:“Hibiden”; manufactured by SUMITOMO PHARMACEUTICALS CO. LTD., Japan))enclosed in said second bag-shaped members (48, 64 (or 68), 88, 108) wasfallen down into the container. As a result, it is now possible that adisinfecting/sterilizing treatment of the inner side of the container(41, 61, 81, 101) including the medium is conducted without detachingthe cover (42, 62, 82, 102) from the container (41, 61, 81, 101).

Example 10

By referring to FIGS. 17 to 33, the following materials were used toconstruct the microorganism-detecting apparatus having the followingsize:

Thus, the size from the bottom of the container (241) of themicroorganism-detecting apparatus shown by FIGS. 17 and 18 to the top ofa cap member (249) thereof (i.e. length of the microorganism-detectingapparatus) was about 201 mm.

Container (241): a hollow cylinder having a bottom (inner diameter:about 9.0 mm; height: about 73 mm) made of polypropylene (thickness:about 1.0 mm) (At the outside near the opening, there were screw threadsfor connection.)

Rod-shaped part (member) (246): made of polypropylene; diameter: about2.5 mm; length: about 77 mm

Microorganism-collecting end (246 b): made of absorbent cotton; an endof “swab” having a maximum diameter of about 5 mm and a length of about15 mm

Cover (243): The hollow cylindrical body (inner diameter: about 13.0 mm;length: about 96 mm; length of the engaging portion (259): about 10 mm)was made of polypropylene (thickness: about 1.0 mm). Inner side of theconnecting part (259) had screw threads.

Partition member (296): a disk with a diameter of about 13 mm made ofpolypropylene (thickness: about 1 mm). At its center a connectingstructure for a rod-shaped member of the microorganism-collecting partwas placed. There were four perforated holes (through holes) in a shapeof a fan. The partition member (296) was engaged with the connectingpart (259) of the cover (243).

Cover (247): This (inner diameter: about 10 mm; length: about 57 mm;length of the head (245) (a connecting part with a cap (249)): about 13mm) was made of polypropylene (thickness: about 1 mm). Inner side of theconnecting part had screw threads. Ridge (or protruding portion) (297)placed on the inner side of the cover (243) (the protruding portionranges to a protrusion (252)) and the groove (299) located at theoutside of the cover (247) were made in such a manner that they wereengaged (or geared) each other whereby, when the cover (247) wasinserted into the cover (243), they were tightly joined.

Partition member (235): This was made of polypropylene (thickness: about1 mm) and its diameter was about 9 mm. There were four perforated holes(through holes) in a shape of a fan. The partition member (235) wasengaged with an end of the side of the container (241) of the cover(247).

Cap material (249): made of polypropylene (thickness: about 1 mm).Diameter of the top side (561) which was opposite to the container (241)was about 15 mm. A cover (562) for a cap member was engaged therewithwhere diameter of the screw thread (257) was about 11 to 13 mm anddiameter of the bottom (256) at the side of the container (241) wasabout 9 mm. The screw threads (257) engaged with the grooves made insideof said connecting part (245). The screw threads formed at the inside ofsaid connecting part (245) had a sufficient width (length of an axialdirection of the cover (247)) so that the cap member (249) was able tobe rotated and pushed thereinto.

First bag-shaped member (244): an ampule (inner diameter: about 9 mm;length: about 43 mm) made of glass (thickness: about 0.5 mm). Itreceived, for example, the improved media as shown in Example 11mentioned later and was made into an apparatus for detectingStaphylococcus aureus, Vibrio parahaemolyticus, Salmonella orEscherichia coli accordingly.

Second bag-shaped member (248): An ampule made of glass (thickness;about 0.5 mm) having inner diameter of about 9 mm and length of about 36mm and receiving a disinfectant/bactericide as shown in Example 7therein.

After manufacturing the microorganism-detecting apparatus having theabove-mentioned constitution, it was sterilized by a sterilizer usingethylene oxide. The sterilization was conducted, for example, at thetemperature of 30 to 50° C. in a gas consisting of about 20% of ethyleneoxide and about 80% of carbon dioxide gas under the pressure of about0.3 kg/m² for about five hours. The apparatus after the sterilizationwas placed in a bag made of polyethylene film (thickness: about 100 μm)evaporated with aluminum for shielding the light and stored untilimmediate before use. A medium of the following Example 11 was used byplacing into the above-mentioned ampule (the first bag-shaped material(244)) followed by sealing.

Similarly were prepared the microorganism-detecting apparatuses byreferring to FIGS. 34 to 51.

Example 11

(Preparation of a Microorganism-Detecting Apparatus Using ImprovedMedium)

(i) For a purpose of taking out from an ampule, a liquid medium issuitable and, from such a viewpoint, an improvement in media wasconducted. Further, with an object of improving the specificity to themicroorganism and of improving the operability of themicroorganism-detecting apparatus, the composition for the medium wasinvestigated whereby there will be no need of using a disk member (7)holding the antibiotics and an improvement was conducted for giving aselectivity to the medium. At the same time, investigation was made onpH indicators as well for such an improvement. As a result, the mediahaving the following compositions were found to be appropriate:

(a) Improved Medium for Salmonella (1) Composition

Base Medium: Tryptone (DIFCO) 5 g Yeast extract (DIFCO) 3 g L-(+)-Lysinemonohydrochloride (WAKO; special grade) 10 g Glucose (anhydrous) (WAKO;special grade) 1 g Sodium chloride (WAKO; special grade) 8 gMonopotassium dihydrogen phosphate (WAKO; sp gr) 1.6 g Sodiumthiosulfate pentahydrate (WAKO; special gr) 0.2 g Ammonium iron(III)citrate (brown) 0.3 g (WAKO; extra pure gr) Magnesium chloridehexahydrate (WAKO; special gr) 20.3 g Bromcresol Purple (WAKO; specialgrade) 0.02 g 0.4% Malachite Green solution 30 ml (Malachite Greenoxalate (WAKO; special grade) (0.4 g) was added to a small amount ofpure alcohol, the mixture was well ground and pure water was addedthereto to make the total volume 100 ml)

(2) Method of Preparation

Each of the components in the above basic medium was weighed and 1000 mlof pure water was added thereto followed by well shaking and theresulting homogeneous floating liquid was mixed with 30 ml of a 0.4%Malachite Green solution followed by dissolving with heating (at 100° C.for 20 minutes). The medium was adjusted to pH 5.3 to 5.7. Aftercooling, the medium was filtered through a filter of 0.2μ.

(b) Improved Medium for Vibrio parahaemolyticus

Base Medium: Salt-polymyxin broth (NISSUI SEIYAKU) 33 g D-(−)-Mannitol(WAKO; special grade) 20 g Sodium citrate dihydrate (WAKO; specialgrade) 8 g Sodium thiosulfate pentahydrate (WAKO; special gr) 0.2 gBromocresol Purple (WAKO; special grade) 0.02 g

(2) Method of Preparation

Each of the components for the above basic medium was weighed and 1000ml of pure water was added thereto followed by well shaking to prepare ahomogeneous floating liquid. The medium was adjusted to pH 7.0 to 7.4and sterilized at 121° C. for 15 minutes. Immediately aftersterilization, it was cooled rapidly.

(c) Improved Medium for Escherichia coli Group

Base Medium: Lauryl tryptose broth (DIFCO) 35.6 g Lactose monohydrate(WAKO; special grade) 5 g Bromthymol Blue (WAKO; special grade) 0.04 g

(2) Method of Preparation

Each of the components for the above basic medium was weighed and 1000ml of pure water was added thereto followed by well shaking to prepare ahomogeneous floating liquid. The medium was adjusted to pH 6.75 to 7.25and sterilized at 121° C. for 15 minutes. Immediately aftersterilization, it was cooled rapidly.

(d) Improved Medium for Staphylococci

Basic Medium: Tryptone (DIFCO) 10 g Yeast extract (DIFCO) 5 gD-(−)-Mannitol (WAKO; special grade) 10 g Dipotassium monohydrogenphosphate (WAKO; sp gr) 5 g Lithium chloride monohydrate (WAKO; specialgrade) 5.5 g Glycine (WAKO; special grade) 16.5 g Sodium pyruvate (WAKO;special grade) 12 g Phenol Red (WAKO; special grade) 0.025 g 1% Aqueoussolution of potassium tellurite 15 ml Potassium tellurite (WAKO) 1 g(Pure water (100 ml) was added to 1 g of potassium tellurite and themixture was dissolved)

(2) Method of Preparation

Each of the components in the above basic medium was weighed and 1000 mlof pure water was added thereto followed by well shaking to prepare ahomogeneous floating liquid. The medium was adjusted to pH 7.25 to 7.75and sterilized at 121° C. for 15 minutes. When it was cooled down to 50°C. or lower, 15 ml of 1% aqueous solution of potassium tellurite wasadded. After well mixing, the mixture was sterilized by filteringthrough a filter of 0.2μ.

(ii) Measurement of Microorganism-Detecting Sensitivity and Specificityfor Microorganism of Each Medium

Control strains, i.e. Salmonella (amount: 10⁹ cells), Vibrioparahaemolyticus (amount: 10⁹), Escherichia coli group (amount: 10⁹) andStaphylococcus (amount: 10⁸), were dispersed in 1.5 ml of each medium tomake the initial amount as shown in Tables 8 to 11 followed byincubating at 37° C. for 24 hours in air.

Growth of each microorganism was judged by means of changes in colorafter the incubation. Incidentally, all of the above-mentioned controlstains were the clinically separated strains and were identified by API20 E (trade name; bioMerieux-Vitek Japan, Ltd., Japan) for Escherichiacoli, Salmonella and Vibrio and by API Staph (trade name;bioMerieux-Vitek Japan, Ltd., Japan) for Staphylococcus.

In API 20 E (trade name; bioMerieux-Vitek Japan, Ltd., Japan), thefollowing is exemplified in a positive rate table under the incubatingconditions of 35 to 37° C. for 24 or 48 hours:

They are Escherichia coli 1 and Escherichia coli 2 for Escherichia coli;Salmonella arizonae, Salmonella choleraesuis, Salmonella paratyphi A,Salmonella spp. and Salmonella typhi for Salmonella; and Vibrioalginolyticus, Vibrio cholerae, Vibrio hollisae, Vibrio metschnikovii,Vibrio mimicus, Vibrio parahaemolyticus and Vibrio vulnificus forVibrio.

In API Staph (trade name; bioMerieux-Vitek Japan, Japan), the followingis exemplified in a positive rate table under incubating condition of 35to 37° C. for 18 to 24 hours for Staphylococcus:

They are Staphylococcus aureus, Staphylococcus auriculans,Staphylococcus capitis, Staphylococcus caprae, Staphylococcus camosus,Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcusepidermidis, Staphylococcus haemolyticus, Staphylococcus hominis,Staphylococcus hyicus, Staphylococcus lentus, Staphylococcuslugdunensis, Staphylococcus saprophyticus, Staphylococcus schleiferi,Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneriand Staphylococcus xylosus.

TABLE 7 (unit: CFU/ml) Microorganism- Sensitivity Detecting ApparatusBefore Improvement After Improvement Kit for Staphylococcus 3.0 × 10³1.9 × 10² Kit for Salmonella 3.0 × 10⁴ 1.4 × 10² Kit for Vibrio 3.0 ×10⁵ 3.1 × 10² parahaemolyticus Kit for Escherichia 3.0 × 10⁴ 1.5 × 10²coli group

It is understood from Table 7 that, in the improved media, improvementin sensitivity was achieved in all microorganisms. A significantimprovement was achieved especially in Vibrio parahaemolyticus. Thefollowing Tables 8 to 11 show the result of judgment of each of themedia for the improved ones having the above-mentioned compositions ascompared with the media before the improvement. In the tables, “FoodStamp” is a conventional name for a device for detecting microorganisms,for example, available from EIKEN KIZAI CO., LTD., Japan (trade name:“PETAN CHECK”), DENKA SEIKEN K. K., Japan (trade name: “DD CHECKER“SEIKEN””) and NISSUI PHARMACEUTICAL CO., LTD (trade name: “SHOKUZAICHECK “NISSUI””), and “San Coli” is a trade name for “Coli formsDetection Paper” available from SAN KAGAKU K. K., Japan.

TABLE 8 Kit for Staphylococcus Rate : 10 × −3 −4 −5 −6 −7 −8 DilutionAmount of 10⁵ 10⁴ 10³ 10² 10¹ 10⁰ Microorganism Media before Change inColor (+) (+) (+) (±) (−) (−) Improvement Colony Numbers NumerousNumerous Numerous Negative Negative Negative Media after Change in Color(+) (+) (+) (+) (−) (−) Improvement Colony Numbers Numerous NumerousNumerous Numerous Negative Negative Food Stamp Numerous NumerousNumerous Negative Negative Negative “San-Coli” (−) (−) (−) (−) (−) (−)

TABLE 9 Kit for Salmonella Rate : 10 × −3 −4 −5 −6 −7 −8 Dilution Amountof 10⁶ 10⁵ 10⁴ 10³ 10² 10¹ Microorganism Media before Change in Color(+) (+) (+) (−) (−) (−) Improvement Colony Numbers Numerous NumerousNumerous Negative Negative Negative Media after Change in Color (+) (+)(+) (+) (+) (−) Improvement Colony Numbers Numerous Numerous NumerousNumerous Numerous Negative Food Stamp Numerous Numerous NumerousNegative Negative Negative “San-Coli” (−) (−) (−) (−) (−) (−)

TABLE 10 Kit for Vibrio parahaemolyticus Rate : 10 × −3 −4 −5 −6 −7 −8Dilution Amount of 10⁶ 10⁵ 10⁴ 10³ 10² 10¹ Microorganism Media beforeChange in Color (+) (+) (−) (−) (−) (−) Improvement Colony NumbersNumerous Negative Negative Negative Negative Negative Media after Changein Color (+) (+) (+) (+) (+) (−) Improvement Colony Numbers NumerousNumerous Numerous Numerous Numerous Negative Food Stamp NumerousNumerous Numerous Negative Negative Negative “San-Coli” (−) (−) (−) (−)(−) (−)

TABLE 11 Kit for Escherichia coli group Rate : 10 × −3 −4 −5 −6 −7 −8Dilution Amount of 10⁶ 10⁵ 10⁴ 10³ 10² 10¹ Microorganism Media beforeChange in Color (+) (+) (+) (−) (−) (−) Improvement Colony NumbersNumerous Numerous Numerous Negative Negative Negative Media after Changein Color (+) (+) (+) (+) (+) (−) Improvement Colony Numbers NumerousNumerous Numerous Numerous Few Negative Food Stamp Numerous NumerousNumerous Negative Negative Negative “San-Coli” (−) (−) (−) (−) (−) (−)

Staphylococcus aureus (MRSA), Staphylococcus aureus (MSSA),Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcushaemolyticus, Enterococcus sp., Bacillus sp., Salmonella typhimurium,Salmonella typhi, Proteus vulgaris, Proteus mirabilis, Citrobacterfreundii, Citrobacter diversus, Klebsiella pneumoniae, Klebsiellaoxytoca, Enterobacter aerogenes, Enterobacter closcae, Serratiamarcescens, Hafnia alvei, Morganella morganii, Vibrio parahaemolyticus,Vibrio fluvialis, Vibrio vulnificus, Aeromonas sp., Plesiomonasshigelloides, Escherichia coli (O-157 EHRC), Escherichia coli (O-55EPEC), Escherichia coli (O-124 EIEC), Escherichia coli (O-25 ETRC),Pseudomonas sp., Acinetobacter Sp., Flavobacterium sp., etc. were usedand their detection was conducted whereupon good results were obtained.Relation between the microorganisms showing positive result and thechanges in color of the medium is given in Table 12. It was noted thatcoloration was good and the resulting color had an excellentdiscriminating ability.

TABLE 12 Microorganism showing Positive Reaction and Color ChangeMicroorganism- Detecting Microorganisms showing Before Improvement AfterImprovement Apparatus Positive Reactions Negative Positive NegativePositive Kit for Staphylococcus aureus pink (weak) orange red yellowDetecting (strong) yellow Staphylococcus Others pink yellow red Kit forSalmonella purple dark purple blue purple Detecting Others generaCitrobacter and purple yellow blue purple Salmonella Enterobacter generaKlebsiella, Proteus purple yellow blue and E. coli group Kit for Vibrioparahaemolyticus bluish purple yellowish green purple yellow VibrioOthers genera Enterococcus bluish purple yellow purple yellow parahaemo-and Serratia lyticus genera Klebsiella, Proteus bluish purple yellowpurple and Citrobacter Kit for Escherichia coli group purple yellowgreen yellow Escherichia Others purple yellow or white green coli Group(transparent) Others: refer to the above-mentioned test microorganismsNegative: the same color as before the test

An improved medium for Escherichia coli group mentioned in the aboveExample 11(i)(c) where Bromcresol Purple was substituted for BromthymolBlue gave a good result in detecting the microorganisms in food and foodmaterials. For example, a good result was obtained in the detection of amicroorganism in milk products by the use of a microorganism-detectingapparatus (containing the improved medium for Escherichia coli)constituted according to Example 10 (particularly by referring to FIGS.34 to 51). The coloration was good and the resulting color had anexcellent discriminating ability.

Example 12

By utilizing a microorganism-detecting apparatus as shown in FIG. 35wherein a medium suitable for measuring numbers of viable cells is used,target microorganisms are cultured. After incubation for a determinedperiod, the cultured medium is measured for turbidity. In theexperimentation, it is checked whether or not the number of viablemicroorganism cells prior to the incubation can be estimated from theresultant turbidity data.

(1) To a medium for measuring the number of viable cells was added asolution of microorganisms with a predetermined concentration (10⁸ to10¹ cells) and the medium was incubated at 37° C. for a certain time (3,5, and 6 hours, respectively).

Medium for measuring the number of viable cells Composition per 1 literof medium Meat extract 3.0 g Casein peptone 15.0 g Yeast extract 5.0 gGlucose 1.0 g

(2) The cultured medium was diluted with a physiological saline solutionto a 2-fold dilution. Then, said 2-fold diluted medium was applied to aterbidimeter (such as a device designed to measure the bacterial densityin a liquid medium), McFarland turbidimeter (available frombioMerieux-Vitek Japan, Ltd., Japan; ATB1550) and measured forturbidity. The results are shown in FIGS. 71 and 72. FIG. 71 is therelation between the number of viable Staphylococcus aureus cells andthe measured turbidity data. FIG. 72 is the relation between the numberof viable Escherichia coli cells and the measured turbidity data.

In this experimentation, the number of the viable cells ranges from 10³to 10⁸ cells after incubation for 5 to 6 hours and it is confirmed thatthere is a proportional relationship between the number of viable cellsand turbidity data. Thus, it is understood that it is possible toestimate the number of viable microorganism cells prior to incubation byutilizing the microorganism-detecting apparatus according to the presentinvention.

Further, when a suitable incubation time and turbidity is selected, itwould enable a person to estimate the the number of viable microorganismcells prior to incubation. A concentration of medium for measuring thenumber of viable cells may be suitably selected in order to give asuitable turbidity value.

While the best mode for carrying out the invention has been described indetail, those familiar with the art to which this invention relates willrecognize various alternative designs and embodiments for practicing theinvention as defined by the following claims.

What is claimed is:
 1. An apparatus for detecting a microorganism,comprising: a container to hold both the microorganism and a medium forculturing the microorganism during an incubation period of themicroorganism; a microorganism-collecting part, locatable within saidcontainer, to collect the microorganism and then transfer themicroorganism into said container; a first member to hold the medium andthen release the medium such that the medium is deposited into saidcontainer and into contact with the microorganism-collecting part duringthe incubation period; and a second member to hold a disinfectant andthen release the disinfectant such that the disinfectant is depositedinto said container and into contact with the medium and themicroorganism after the incubation period.
 2. The apparatus according toclaim 1, wherein said first member comprises a first vessel and saidsecond member comprises a second vessel.
 3. The apparatus according toclaim 1, wherein said first member is to release the medium by applyinga first external force to said first member and said second member is torelease the disinfectant by applying a second external force to saidsecond member.
 4. The apparatus according to claim 1, wherein said firstmember is to release the medium by applying an external force to saidfirst member.
 5. The apparatus according to claim 1, wherein saidcontainer is open at one end and said microorganism-collecting part islocatable within said container by being passed through the open end ofsaid container, and further comprising a medium for culturing themicroorganism contained within said first member and a disinfectantcontained within said second member.
 6. The apparatus according to claim5, and further comprising an antibiotic substance to be added to saidmedium during the incubation period.
 7. The apparatus according to claim6, wherein said antibiotic substance is located within said containerprior to release of the medium from said first member.
 8. The apparatusaccording to claim 7, wherein said first member is to release saidmedium by applying an external force to said first member.
 9. Theapparatus according to claim 8, wherein said second member is to releasesaid disinfectant by applying an external force to said second member.10. The apparatus according to claim 7, wherein said antibioticsubstance is located within said container prior to release of themedium from the first member by one of being located at a discretelocation within said container, being located on saidmicroorganism-collecting member, and being coated on an inner wall ofsaid container.
 11. The apparatus according to claim 10, wherein saidmicroorganism-collecting member comprises a rod and an end memberconnected to an end of said rod, and said antibiotic substance islocated on said microorganism-collecting member by being located on saidend member.
 12. The apparatus according to claim 6, wherein saidantibiotic substance is to be added to said medium during saidincubation period by being mixed with said medium in said first member,such that upon release of said medium from said first member saidantibiotic substance is also released from said first member.
 13. Theapparatus according to claim 5, wherein said medium includes a substancecapable of changing colors in response to growth of the microorganism.14. The apparatus according to claim 5, wherein said medium forculturing the microorganism is selected from the group consisting of:(a) a medium for culturing Salmonella having a composition substantiallycontaining 3 to 7 grams of tryptone, 1 to 6 grams of a yeast extract, 5to 15 grams of lysine, 0.5 to 2 grams of glucose, 7 to 9 grams of sodiumchloride, 1 to 2 grams of monopotassium dihydrogen phosphate, 0.1 to 0.3grams of sodium thiosulfate, 0.2 to 0.4 grams of ammonium iron citrate,15 to 25 grams of magnesium chloride, 27 to 33 milliliters of a 0.4%Malachite Green solution, and 0.01 to 0.03 grams of Bromcresol Purpleper 1,000 milliliters of the medium, with the pH of the medium beingabout 5.3 to 5.7; (b) a medium for culturing Vibrio parahaemolyticushaving a composition substantially containing 15 to 25 grams ofmannitol, 5 to 10 grams of sodium citrate, 0.1 to 0.3 grams of sodiumthiosulfate, 0.01 to 0.03 grams of Bromocresol Purple per 1,000milliliters of the medium, and 25 to 40 grams of a salt polymyxin brothcontaining a yeast extract, peptone, sodium chloride and polymyxin B,with the pH of the medium being about 7.0 to 7.4; (c) a medium forculturing Escherichia coli group having a composition substantiallycontaining 3 to 8 grams of lactose, 0.03 to 0.05 grams of BromthymolBlue or Bromocresol Purple per 1,000 milliliters of the medium, and 26to 43 grams of a lauryl sulfate broth containing tryptose, lactose,monopotassium dihydrogen phosphate, dipotassium monohydrogen phosphate,sodium chloride and sodium lauryl sulfate, with the pH of the mediumbeing about 6.75 to 7.25; and (d) a medium for culturing Staphylococcushaving a composition substantially containing 5 to 15 grams of tryptone,2 to 8 grams of a yeast extract, 5 to 15 grams of mannitol, 2 to 8 gramsof dipotassium monohydrogen phosphate, 5 to 6 grams of lithium chloride,12 to 20 grams of glycine, 10 to 14 grams of sodium pyruvate, 12 to 18milliliters of a 1% aqueous solution of potassium tellurite, and 0.02 to0.03 grams of Phenol Red per 1,000 milliliters of the medium, with thepH of the medium being about 7.25 to 7.75.
 15. The apparatus accordingto claim 5, wherein said first member and said second member are spacedfrom each other, and further comprising a cover for housing said firstmember and said second member, wherein said housing is attachable tosaid container, and wherein said first member is to release said mediumby applying a first external force to said first member and said secondmember is to release said disinfectant by applying a second externalforce to said second member.
 16. The apparatus according to claim 1, andfurther comprising a cover attachable to said container, wherein saidfirst member comprises first a vessel contained within said cover. 17.The apparatus according to claim 16, and further comprising a perforatedplate located between said first vessel and said container when saidcover is attached to said container.
 18. The apparatus according toclaim 17, and further comprising a guide member located between saidperforated plate and said container when said cover is attached to saidcontainer, wherein said guide member is to guide the medium releasedfrom said first vessel into said container.
 19. The apparatus accordingto claim 1, wherein said apparatus is to detect a microorganism selectedfrom the group consisting of enteropathogenic Escherichia coli,Staphylococcus aureus, Vibrio parahaemolyticus and Salmonella.
 20. Theapparatus according to claim 1, and further comprising a coverattachable to said container, wherein said second member is containedwithin said cover such that the disinfectant is released from saidsecond member when said cover is attached to said container.
 21. Theapparatus according to claim 20, wherein said container is transparent.22. The apparatus according to claim 1, wherein said first membercomprises a first vessel, and further comprising a cover attachable tosaid container, with said first vessel located within said cover. 23.The apparatus according to claim 1, wherein said second member comprisesa second vessel, and further comprising a cover attachable to saidcontainer, with said second vessel located within said cover.
 24. Theapparatus according to claim 1, wherein said first member comprises afirst vessel and said second member comprises a second vessel, andfurther comprising a cover to support and house said first vessel andsaid second vessel.
 25. The apparatus according to claim 24, whereinsaid cover comprises a first portion and a second portion, with saidfirst vessel located within said first portion and said second vessellocated within said second portion.
 26. The apparatus according to claim1, and further comprising a first cover portion housing said firstmember and a second cover portion housing said second member, with saidfirst cover portion and said second cover portion being slidablerelative to one another, and further comprising structure on said firstcover portion and said second cover portion to prevent unwanted slidingof said first cover portion relative to said second cover portion. 27.The apparatus according to claim 26, wherein said structure comprises atleast one projection carried by one of said first cover portion and saidsecond cover portion and at least one slot in the other of said firstcover portion and said second cover portion, with said at least oneprojection being receivable within said at least one slot.
 28. Theapparatus according to claim 26, and further comprising a cap memberslidably attached to said second cover portion and structure on saidcover member and said second cover portion to prevent unwanted slidingof said second cover portion relative to said cap member.
 29. Theapparatus according to claim 28, wherein said structure comprises atleast one projection carried by one of said second cover portion andsaid cap member and at least one slot in the other of said second coverportion and said cap member, with said at least one projection beingreceivable within said at least one slot.
 30. An apparatus for detectinga microorganism, comprising: a first member having a first perforatedpartition to receive and hold a first vessel containing a disinfectant;a second member having a second perforated partition to receive and holda second vessel containing a medium, with said second member beingattachable to a container and said first member being slidablyattachable to said second member; and a cap member movably attached to afirst end of said first member; such that the second vessel is broken bysliding said first member relative to said second member whereby themedium is released from said second vessel and deposited into thecontainer during an incubation period, and the first vessel is broken bymoving said cap member relative to said first member whereby thedisinfectant is released from said first vessel and deposited into thecontainer after the incubation period.
 31. The apparatus according toclaim 30, and further comprising a second vessel containing a medium forculturing the microorganism, wherein said medium is selected from thegroup consisting of: (a) a medium for culturing Salmonella having acomposition substantially containing 3 to 7 grams of tryptone, 1 to 6grams of a yeast extract, 5 to 15 grams of lysine, 0.5 to 2 grams ofglucose, 7 to 9 grams of sodium chloride, 1 to 2 grams of monopotassiumdihydrogen phosphate, 0.1 to 0.3 grams of sodium thiosulfate, 0.2 to 0.4grams of ammonium iron citrate, 15 to 25 grams of magnesium chloride, 27to 33 milliliters of a 0.4% Malachite Green solution, and 0.01 to 0.03grams of Bromcresol Purple per 1,000 milliliters of the medium, with thepH of the medium being about 5.3 to 5.7; (b) a medium for culturingVibrio parahaemolyticus having a composition substantially containing 15to 25 grams of mannitol, 5 to 10 grams of sodium citrate, 0.1 to 0.3grams of sodium thiosulfate, 0.01 to 0.03 grams of Bromocresol Purpleper 1,000 milliliters of the medium, and 25 to 40 grams of a saltpolymyxin broth containing a yeast extract, peptone, sodium chloride andpolymyxin B, with the pH of the medium being about 7.0 to 7.4; (c) amedium for culturing Escherichia coli group having a compositionsubstantially containing 3 to 8 grams of lactose, 0.03 to 0.05 grams ofBromthymol Blue or Bromocresol Purple per 1,000 milliliters of themedium, and 26 to 43 grams of a lauryl sulfate broth containingtryptose, lactose, monopotassium dihydrogen phosphate, dipotassiummonohydrogen phosphate, sodium chloride and sodium lauryl sulfate, withthe pH of the medium being about 6.75 to 7.25; and (d) a medium forculturing Staphylococcus having a composition substantially containing 5to 15 grams of tryptone, 2 to 8 grams of a yeast extract, 5 to 15 gramsof mannitol, 2 to 8 grams of dipotassium monohydrogen phosphate, 5 to 6grams of lithium chloride, 12 to 20 grams of glycine, 10 to 14 grams ofsodium pyruvate, 12 to 18 milliliters of a 1% aqueous solution ofpotassium tellurite, and 0.02 to 0.03 grams of Phenol Red per 1,000milliliters of the medium, with the pH of the medium being about 7.25 to7.75.58.
 32. The apparatus according to claim 30, wherein said capmember is movably attached to said end of said first member by beingrotatably attached to said end of said first member, and the firstvessel is broken by rotating said cap member relative to said firstmember.
 33. The apparatus according to claim 30, wherein said cap memberis movably attached to said end of said first member by being slidablyattached to said end of said first member, and the first vessel isbroken by sliding said cap member relative to said first member, andfurther comprising structure on said first member and said cap member toprevent sliding of said cap member relative to said first member untilafter the second vessel has been broken by sliding said first memberrelative to said second member.
 34. The apparatus according to claim 33,wherein said structure comprises at least one projection carried by oneof said first member and said cap member and at least one slot in theother of said first member and said cap member, with said at least oneprojection being receivable within said at least one slot.
 35. Theapparatus according to claim 30, and further comprising structure onsaid first member and said second member to prevent unwanted sliding ofsaid first member relative to said second member.
 36. The apparatusaccording to claim 35, wherein said structure comprises at least oneprojection carried by one of said first member and said second memberand at least one slot in the other of said first member and said secondmember, with said at least one projection being receivable within saidat least one slot.
 37. A method for quantitatively measuring the amountof viable microorganism cells in a sample, comprising: inserting asample collected with a microorganism collecting part into a container;associating with said container a first member containing a culturingmedium, and a second member containing a disinfectant; releasing saidculturing medium from said first member onto said sample during anincubation period; and releasing said disinfectant from said secondmember onto said sample after said incubation period.
 38. The methodaccording to claim 37, wherein the inserting includes inserting themicroorganism collecting part along with said sample into saidcontainer, the associating includes attaching to an open end of saidcontainer a cover containing said first member and said second member,the releasing of said culturing medium includes applying a firstexternal force to said first member, and the releasing of saiddisinfectant includes applying a second external force to said secondmember.
 39. The method according to claim 38, wherein said first memberis a first vessel and said second member is a second vessel, and whereinthe applying of the first external force breaks said first vessel andthe applying of the second external force breaks said second vessel. 40.The method according to claim 37, and further comprising adding anantibiotic substance to said sample during said incubation period. 41.The method according to claim 37, wherein the viable microorganism cellsto be quantitatively measured are of a microorganism selected from thegroup consisting of enteropathogenic Escherichia coli, Staphylococcusaureus, Vibrio parahaemolyticus and Salmonella.
 42. The method accordingto claim 41, wherein said culturing medium is selected from the groupconsisting of: (a) a medium for culturing Salmonella having acomposition substantially containing 3 to 7 grams of tryptone, 1 to 6grams of a yeast extract, 5 to 15 grams of lysine, 0.5 to 2 grams ofglucose, 7 to 9 grams of sodium chloride, 1 to 2 grams of monopotassiumdihydrogen phosphate, 0.1 to 0.3 grams of sodium thiosulfate, 0.2 to 0.4grams of ammonium iron citrate, 15 to 25 grams of magnesium chloride, 27to 33 milliliters of a 0.4% Malachite Green solution, and 0.01 to 0.03grams of Bromcresol Purple per 1,000 milliliters of the medium, with thepH of the medium being about 5.3 to 5.7; (b) a medium for culturingVibrio parahaemolyticus having a composition substantially containing 15to 25 grams of mannitol, 5 to 10 grams of sodium citrate, 0.1 to 0.3grams of sodium thiosulfate, 0.01 to 0.03 grams of Bromocresol Purpleper 1,000 milliliters of the medium, and 25 to 40 grams of a saltpolymyxin broth containing a yeast extract, peptone, sodium chloride andpolymyxin B, with the pH of the medium being about 7.0 to 7.4; (c) amedium for culturing Escherichia coli group having a compositionsubstantially containing 3 to 8 grams of lactose, 0.03 to 0.05 grams ofBromthymol Blue or Bromocresol Purple per 1,000 milliliters of themedium, and 26 to 43 grams of a lauryl sulfate broth containingtryptose, lactose, monopotassium dihydrogen phosphate, dipotassiummonohydrogen phosphate, sodium chloride and sodium lauryl sulfate, withthe pH of the medium being about 6.75 to 7.25; and (d) a medium forculturing Staphylococcus having a composition substantially containing 5to 15 grams of tryptone, 2 to 8 grams of a yeast extract, 5 to 15 gramsof mannitol, 2 to 8 grams of dipotassium monohydrogen phosphate, 5 to 6grams of lithium chloride, 12 to 20 grams of glycine, 10 to 14 grams ofsodium pyruvate, 12 to 18 milliliters of a 1% aqueous solution ofpotassium tellurite, and 0.02 to 0.03 grams of Phenol Red per 1,000milliliters of the medium, with the pH of the medium being about 7.25 to7.75.
 43. A method for quantitatively measuring the amount of viablemicroorganism cells in a sample, comprising: inserting a sample into acontainer; depositing a culturing medium onto said sample during anincubation period by breaking a first vessel containing said culturingmedium, wherein said first vessel is contained within a first memberattached to an open end of said container with a first perforated memberpositioned between said first vessel and said sample, such that whensaid first vessel is broken said culturing medium flows through saidfirst perforated member and into contact with said sample while thebroken first vessel is prevented by said first perforated member frombeing deposited onto said sample; and after said incubation period,depositing a disinfectant onto said sample by breaking a second vesselcontaining said disinfectant, wherein said second vessel is containedwithin a second member attached to said first member with a secondperforated member positioned between said second vessel and said sample,such that when said second vessel is broken said disinfectant flowsthrough said second perforated member and into contact with said samplewhile the broken second vessel is prevented by said second perforatedmember from being deposited onto said sample.
 44. The method accordingto claim 43, wherein said second member is slidably attached to saidfirst member and a cap member is movably attached to said second member,such that the breaking of said first vessel results from sliding saidsecond member relative to said first member and the breaking of saidsecond vessel results from moving said cap member relative to saidsecond member.
 45. The method according to claim 44, wherein the movingof said cap member includes rotating said cap member relative to saidsecond member.
 46. The method according to claim 44, wherein the movingof said cap member includes sliding said cap member relative to saidsecond member.
 47. The method according to claim 43, and furthercomprising adding an antibiotic substance to said sample during saidincubation period.
 48. The method according to claim 43, wherein theviable microorganism cells to be quantitatively measured are of amicroorganism selected from the group consisting of enteropathogenicEscherichia coli, Staphylococcus aureus, Vibrio parahaemolyticus andSalmonella.
 49. The method according to claim 48, wherein said culturingmedium is selected from the group consisting of: (a) a medium forculturing Salmonella having a composition substantially containing 3 to7 grams of tryptone, 1 to 6 grams of a yeast extract, 5 to 15 grams oflysine, 0.5 to 2 grams of glucose, 7 to 9 grams of sodium chloride, 1 to2 grams of monopotassium dihydrogen phosphate,
 0. 1 to 0.3 grams ofsodium thiosulfate, 0.2 to 0.4 grams of ammonium iron citrate, 15 to 25grams of magnesium chloride, 27 to 33 milliliters of a 0.4% MalachiteGreen solution, and 0.01 to 0.03 grams of Bromcresol Purple per 1,000milliliters of the medium, with the pH of the medium being about 5.3 to5.7; (b) a medium for culturing Vibrio parahaemolyticus having acomposition substantially containing 15 to 25 grams of mannitol, 5 to 10grams of sodium citrate, 0.1 to 0.3 grams of sodium thiosulfate, 0.01 to0.03 grams of Bromocresol Purple per 1,000 milliliters of the medium,and 25 to 40 grams of a salt polymyxin broth containing a yeast extract,peptone, sodium chloride and polymyxin B, with the pH of the mediumbeing about 7.0 to 7.4; (c) a medium for culturing Escherichia coligroup having a composition substantially containing 3 to 8 grams oflactose, 0.03 to 0.05 grams of Bromthymol Blue or Bromocresol Purple per1,000 milliliters of the medium, and 26 to 43 grams of a lauryl sulfatebroth containing tryptose, lactose, monopotassium dihydrogen phosphate,dipotassium monohydrogen phosphate, sodium chloride and sodium laurylsulfate, with the pH of the medium being about 6.75 to 7.25; and (d) amedium for culturing Staphylococcus having a composition substantiallycontaining 5 to 15 grams of tryptone, 2 to 8 grams of a yeast extract, 5to 15 grams of mannitol, 2 to 8 grams of dipotassium monohydrogenphosphate, 5 to 6 grams of lithium chloride, 12 to 20 grams of glycine,10 to 14 grams of sodium pyruvate, 12 to 18 milliliters of a 1% aqueoussolution of potassium tellurite, and 0.02 to 0.03 grams of Phenol Redper 1,000 milliliters of the medium, with the pH of the medium beingabout 7.25 to 7.75.